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My Teaching: TAing Experimental Biochemistry

1. any information that I need to get to students (or would like to make available to students) right now
2. any comments about the course that I wish to make
3. a HTMLized version of the intro sheet that I normally give out at the beginning of each semester.

Information for Current Students

The statistical workup for all sections is due 9/16/05. Next week's lecture will be discussing how to determine outliers and whether you need to redo the calculation with outliers removed.

9/8/05:
I am hearing that some people are having some difficulties with the statistical workup. Consult Dr. Chase or Dr. Kahn (room 120) on it first (and Dr. Chase will be lecturing on it next week), but if they aren't available for some reason, I will try to help - drop by or email me to make an appointment.

9/10/05:
In regard to turnitin.com, we do want the entire lab reports turned in on paper. (Typed is preferable but not required, as long as we can read what you have written. I particularly prefer lab reports that I grade (electrophoresis and isoelectric focusing) being typed, but again this is not a requirement, and I do not expect you to type out calculations.) Only part of each of the lab reports - varying with each report (ask when the time for a report comes around), but including any answers to questions. (Don't include the text of the questions themselves in what you submit to turnitin.com! Also do not include numerical material that will be the same between members of a lab group - as in don't just submit the entire lab report; this will result in "false positive" plagarism reports that are irritating to deal with.) Note that, as well as working independently (of each other) on the questions, we expect you to properly cite any sources that you use (other than the lab manual, lecture material, or similar - ask if you are uncertain). For information on how to get and use a turnitin.com account, talk to Rob Muldowney in Lipman Hall 202 (or email him at robertm@beatrice.rutgers.edu or muldowney@aesop.rutgers.edu). We will be giving out the section numbers and passwords in the lab when we come to the first lab for which you will need to turn something in to turnitin.com (namely, the questions in the pH lab).

9/14/05:
We have wound up sending out the turnitin.com section numbers and passwords to y'all by email. Please let us know immediately if you didn't get said email (title of "My webpage; section #s and passwords for turnitin.com")!

9/16/05:
I will be here and available for help on the pH lab report for most of Sunday and Monday afternoon and evening. If you are coming some distance and need to make sure that I will be in when you come by - especially on Sunday, when you may have problems getting into the building - email me first. (I will probably go out at some point to get some food/etcetera on both days, and am currently feeling not at my best due to a sinus infection and may have to go upstairs to Lipman Hall 325 to lie down at times. I am thus not sure exactly when I will be available on Sunday and Monday. If I am out, I will try to remember to leave a note on the door of 119 as to when I will be back and/or where I am.)

9/17/05:
I am getting some questions on how formal the writeup for the pH lab needs to be - whether you need to do an abstract, have the procedure et al written up, etcetera. No, you don't need to put down anything not mentioned in the section "The Report" in the lab manual, unless specifically told otherwise. (If you had a problem during the procedure and as a result are uncertain on something, do mention that - otherwise, you don't need to go over the procedure.) If you had any uncertainty in figuring out which unknown you had, do mention that, and how you decided which one you had. You will also need to answer all the questions in the "The Report" section, or otherwise specifically mentioned as something you need to put in the report, of course. The pH lab is designed to have a short lab report that you can reasonably write up with only a week to work on it, and that we can get back to you before the drop date (as we're supposed to do by University policy - we're supposed to give you feedback before the drop date if at all possible).

9/19/05: You should now be able to submit the pH lab questions to turnitin.com - sorry, I had thought that it was already set up!

What you submit from the pH lab to turnitin.com are the answers to the questions on pages 15 and 16 of the lab manual - not anything else. And you also need to include a copy of this, printed out, with your lab report (along with the data, graphs, filling in the various blanks, etcetera).

In the page of the pH lab (20) with the various blanks to fill in and a question down below, the numbered questions (starting with "1. What is the starting pH?") are all about the titration of the K biphthalate. Question 5 on that page tells you how to calculate the molarity of the "~0.5M" KOH, BTW.

9/20/05:
You should only submit the answers to the questions, not the questions themselves, to turnitin.com. You should not try to submit anything that is not text - no figures/drawings or calculations. You can submit the answers to turnitin.com multiple times, and can revise them each time (e.g., if you forgot to cite something), provided you give us the final copy with your lab report, until the due date - after the due date, you can only submit it to turnitin.com once (and I don't believe you can resubmit it after the due date if you have already submitted it prior to the due date). You must give us the answers to the questions with your lab report also, including any figures/drawings and calculations - the copy with the lab report is what gets graded; we will just check the turnitin.com version for, first, plagarism, and second, for whether it is the same as the version with your lab report. For getting a turnitin.com account or for help using said account, consult Rob Muldowney if at all possible.

9/21/05:
If you are coming by 118 to see me and the door to 119 is locked but a "do not disturb" sign is not present, look through the glass to see if the light is on in my office (at the back on the left). If it is, knock.

9/23/05:
Please remember that, while I am frequently here and available for turning lab reports in (noting that, unless we specify otherwise, weekend days do not count for points off for lateness), the building is locked after ~7 PM on weekdays and all the time on weekends - you will need to have someone with a card/key let you in (note that someone who has a card can also let you into the 202 computer lab, and that the computer lab in 214 is generally unlocked), call me from outside the building so I can let you in, or tap on my window to get my attention so I can let you in - these options are in order of preference. For the latter two, I suggest emailing in advance to make sure I'll be here at the time you will be arriving, so you aren't stuck waiting around outside for me to come back (or come down from upstairs if I'm laying down up there).

9/27/05:
A couple of things:

1. I will be available Sunday afternoon and evening and Monday late afternoon and evening for assistance with lab reports (namely, the first report - without the heavy statistical workup - for the protein lab), although note that I may not be present quite all the time (hopefully, my sinus infection will be better by then, but if not I am likely to need to lay down at times due to medication side effects; moreover, I will need to go out to get something to eat at various points), so especially if it's Sunday or you're coming any significant distance, email or call me first to check to make sure I'll be in.
2. Quite a few people have not submitted their pH lab questions to turnitin.com. Some of these people may not have turned in the lab report paper copy yet, such as many of those in the Friday section, but you do need to get it in ASAP once you get in the paper copy (or vice-versa, but realize that we use the paper copy for grading, so when we get it partially determines when you get the lab reports back!). Please make sure when you submit something to turnitin.com that you submit it in the right category - I found one person who had submitted their pH lab into the folder for the protein lab, which as it happens (due to bugs in turnitin.com's system) is on top in some of the lab sections despite being due later.

Some people appear to have gotten mixed up on the point value of the various labs. The pH lab, and all labs after that (except that I'm not sure about the protein lab due to the seperate statistical analysis), are out of 100 points (with 2 points off from that for each day of lateness, not counting weekends and holidays unless otherwise noted). The resulting points are then multiplied by the number of weeks that the lab involves (e.g., for the enzyme lab (the longest one, at 4 weeks), the points are multipled by 4 after any subtraction for lateness). Only the pipetting/dilution/spectrophotometry lab, or rather the statistical workup thereof, is out of any less points. Note that if any lab is not turned in you cannot pass the course, even if you would actually pass it by your point total despite the 0 points from that lab (which is highly unlikely with regard to the longer labs).

Differences in turnitin.com usage between General Biochemistry tutorials and Experimental Biochemistry lab reports:

• For Experimental Biochemistry, do not include the questions when you put the answers into turnitin.com. (You have to put them in for General Biochem tutorials because the questions aren't numbered, for some idiot reason. This results in lots of false positives from turnitin.com.)
• Rob has things set for General Biochem so that you can't resubmit tutorials without his (or that of someone teaching the course's) permission. For Experimental, you can resubmit them as long as the due date has not passed - you cannot submit again after the due date if you have submitted them prior to the due date, and can only submit them once if you submit them after the due date for the first (and, thus, last) time.

Corrections to the protein lab (besides those on the board in 207, or rather the below are a more readable version of some of the corrections on the board in 207 - look at the board in 207!):

• On page 10, under "Data Analysis", in steps 2 and 3, substitute "protein" for "ovalbumin", since you don't know whether the "unknown protein" is ovalbumin (it usually isn't, but I don't know whether Emilia may have done things differently this time around). She has. It's ovalbumin, unless there was a miscommunication. What varies between people is what concentration it is.
• On page 16, for the second line of tubes for Coomassie, instead of "mg oval" it should be "ml oval" (with the ml above it being the number of mls of interfering substance or of unknown protein).

10/3/05:
For question 1, instead of glycylglycine (one glycine peptide-bonded to another glycine), we are using glycylglycylglycine (one glycine peptide-bonded to another glycine peptide-bonded to a third glycine - so 2 peptide bonds in total; hint: see the top of page 5 for why this would matter).

10/4/05:
In regard to the statistical analysis of the protein lab data, do not use the attached data (on the last page of the statistical appendix) except for practice; just turn in the results for your own data. We will be posting up a copy of the results from the analysis of the attached data so you can check your practice results and see how to do it if you are confused.

Questions 1 and 2 in the protein lab (which should really be combined) ask about which unknown substance is which of the 6 unknown substances, but only name 5 of the unknown substances. The unknown substances are, in semi-reverse-alphabetical order (not A-F order!):

• Tyrosine (This is just free tyrosine floating around in solution, not peptide-bonded to anything)
• RNA
• Glycylglycylglycine
• Protamine sulfate
• Lysozyme
• BSA (Bovine Serum Albumin)

Remember that all of the methods except for the Coomassie Blue will have a 0,0 point, on both the standard curve graph and the unknown graph. The Coomassie Blue will have a 0 mg protein point that will be above 0 for both A₅₉₅/A₄₆₆ and A₅₉₅. You will then copy these points from the standard curves to the unknown curves, since 0 mg ovalbumin = 0 ml unknown protein. (No protein is no protein!)

You do not need to plot the absorbances of the unknown substances or of the interfering substances. You will get a rather weird graph if you do!

You can remove points if you have reason to, such as if the absorbances are going down with increasing amounts of protein (however, this is normal for the Coomassie A₄₆₆), provided that:

• You say that you did so, and what the reason is - ideally, also include a graph showing what the data looks like without removing those points. This is especially important if you have to remove the 0-protein point for some weird reason (check with us before you do so!), since otherwise we may think you didn't know that it should normally be on the graph.
• You leave at least 3 points, ideally including the 0-protein point - preferably, at least 4 points, including the 0-protein point. Only remove further points when you already have only 4 points left due to something really looking bad (ask if unsure), not just because of the R-squared value being low.

For the Coomassie Blue, it is often best to create two standard curves - one not including points with A₅₉₅/A₄₆₆ above 2, and the other including such points (and for which you may wish to take out some of the lowest points - the first point after the 0-protein one is particularly likely to have a A₅₉₅ value that actually goes down a bit, in which case it should probably be removed). Unknown protein samples with a A₅₉₅/A₄₆₆ below 2 should be interpreted using the first standard curve; others should be interpreted using the second standard curve. When you work up the statistical analysis, you may find the A₅₉₅ will work best in some regions of the curve, but for now, just worry about the A₅₉₅/A₄₆₆.

10/5/05:
We now have a policy for what happens if you turn a lab in on a given date, but do not turn it into turnitin.com until after the due date and after the date you turn the paper copy in. It is 1 point per day, except weekends/holidays except as specified otherwise. For example, if your lab is due Tuesday, you turn in the paper copy on Wednesday (losing 2 points for lateness), and you turn in the proper portions to turnitin.com on Friday/Saturday/Sunday, you lose a further 2 points (one for Thursday, one for Friday/Saturday/Sunday). We will, however, make exceptions for computer problems (try to notify me ASAP about such, however!). If anyone does not have the proper section ID and password for turnitin.com, please contact me ASAP for it (including information on which section you are in) so you can turn in the answers to the questions for the pH and protein labs. (Yes, for the turnitin.com results for the protein lab, we will be taking into account that people in a given group will be using the same data. That you tell us what the reasoning is for your answers, and do that seperately and with citations of any sources used, is the important part.)

For anyone (still) wondering how to get the E^{1 mg/mL} for the protein determination procedure, here is the easiest way I know of to do it:

• For the methods in which you have plotted (for the standard curve) the absorbance vs the mg of ovalbumin (all except the UV method), take the slope (which is A/mg) and multiply it by the number of mLs of solution used. (For the E^{1 mg/mL} determination, this should be the total number of mLs of solution prior to taking out 3 mLs to put in the cuvette - e.g., 5 mL for the Biuret method. For figuring up sensitivity, it can be argued that one should instead use the total number of mLs of sample that you can use in a given method; we will be accepting either way of doing it.)
• For the UV method, in which you were asked (for some reason) to plot absorbance vs mg/ml (concentration) of ovalbumin for the standard curve instead of absorbance vs mg, you can just use the slope as the E^{1 mg/mL}, since it is change in absorbance per change in concentration in mg/mL.

Incidentally, for anyone who hasn't had cause/opportunity to notice, I am generally a night person (unless my sleep cycle has rotated to very strange hours for me, which does happen at times). Unless I have a morning appointment (groan!) or wake up and can't get back to sleep (again, groan!), - which is when you may see me in the morning labs - I will generally not be in in the mornings, particularly early mornings - except very early mornings, when I may either have never gone to bed or have simply taken a nap (usually in Lipman Hall 325) and gotten back up to work some more. So trying to make an appointment to see me sometime in the morning is not likely to work (I wouldn't be mentally functional enough to help you even if I was technically awake!). Try afternoon/evening/night.

10/6/05:
Several things:

• Ignore the material in the lab manual about A₂₈₀ being twice A₂₆₀ or the other way around for UV - this is extremely approximate in real life, given errors in absorbance measurements, the presence of other amino acids than tyrosine and tryptophan in proteins, etcetera. Instead, just look for ones with significant UV absorbance at whether the A₂₈₀ or the A₂₆₀ is higher - if it is something with tyrosine or tryptophan, the A₂₈₀ should be higher; if it is a nucleic acid, the A₂₆₀. For things with low UV absorbance, you may find that the A₂₆₀ is actually slightly higher - this can indicate a lack of tyrosines and tryptophans.
• As well as looking at what didn't react in various methods, also look (especially for the UV and Coomassie Blue methods) at what reacted more than an equal amount of ovalbumin.
• Don't just call anything with an absorbance under 0.1 "no reaction" - compare it with the absorbance of an equal amount of ovalbumin. If, for example, an equal amount of ovalbumin gave you an absorbance of 0.099, then 0.088 is probably positive, while 0.009 would be negative.
• My schedule over the weekend is that I will be available most of Sunday and Monday afternoons and evenings/nights, and will post up something on my door as to where I am if I am elsewhere (although you may wish to also check in 214, 207, and 202 to see if I am up there helping other students). Check with me for other times, and be sure to contact me before making a special trip over here, particularly on Saturdays and Sundays or after (a bit before) 7 PM on any day of the week, when the outside doors will be locked.

10/8/05:
(Yes, I'm in, and will be most of the weekend.) Regarding the statistical appendix, I am personally having a lot of problems with section 4 in it, and am advising Dr. Chase to push back the due date on that part until a more comprehensible version is posted/distributed. I am not the one grading this lab, however, so I don't know if this will happen.

10/9/05:
A clarification regarding part 5 of the statistical workup: Use your own data, not the test data. Also: on Kalideograph, instead of putting in "m1+m2*m0;m1=0.9;m2=5;" for a linear fit using the General Fit, you can just put in "m1+(m2*m0);" as the linear fit equation - you do not need to specify the (starting) parameters. (Once you have in a General Fit equation, you can then go to Curve Fit, under that to General Fit (IIRC), then to the equation you have put in (e.g., "Linear Fit").)

10/10/05:
OK, I talked to Dr. Chase; part 4 of the statistical appendix is now delayed in due date (not sure exactly by how much - it will depend on our conferencing with Dr. Kahn - but almost certainly till after the General Biochem exam on Wednesday, at the minimum). Parts 1-3 and part 5 are still due this Tuesday/Wednesday/Friday.

Parts 1-3 and part 5 are due Friday of this week for all sections. (Sorry to the Friday section, but you're already not been distracted by the statistical appendix work from studying for the General Biochem exam, as Dr. Chase pointed out to me...) Currently, part 4 is likewise due Friday of this week for all sections, provided that Dr. Chase is able to get together a worked-out example version (both of what he had originally intended and of what Dr. Kahn wrote out, the latter of which is what's currently in the statistical appendix) for tomorrow morning's lecture.

10/12/05:
Four things for the statistical workup:

• It is due (including part 4) Friday of this week. If you can get it to me over the weekend, that will also count as in on time - but keep in mind the building is locked after ~7:00 PM on all days, and all day on Saturday and Sunday.
• You are not expected to upload any of the statistical workup to turnitin.com - it has entirely too many calculations and the numbers would be entirely the same for everyone in a lab group. (I will be checking turnitin.com this afternoon/evening to see how things are going on there.)
• For part 5, the "line value - observed" absorbance is otherwise known as a "residual" (or the residual may be the reverse, in which case just multiply by negative 1 to get this). Most statistical programs will tell you the residuals if you ask for them - I know this is true of Sigmaplot (in doing the Regression Wizard, it asks you for whether to put the residuals on the spreadsheet and, if so, the column in which to put them; these are also in the Report that it gives), and suggest looking for checkboxes in Excel, etcetera.
• I will be available for help on the statistical workup Saturday and Sunday afternoon/evening (but email/call first to make sure I haven't needed to go out or go upstairs to lie down), and may be available at other times - drop by my office if the building is open, or email me if it isn't.

Good luck on the General Biochem exam! I will be proctoring over in Lipman today (for people who - like me - have ADD/ADHD and have problems with distractions) and grading tomorrow (and maybe tonight).

10/14/05:
With regard to the statistical workup, a summary (sorry about any lack of readability!) for part 4 (and a bit on part 3) is on the whiteboard in 206/207 (whichever one is the room we're allowed to eat in - not the lab itself). Also, be sure to get a copy of the revisions for part 4 that Dr. Chase handed out in lecture! These take priority over whatever it says in the statistical appendix for part 4.

You only need to do the residuals (for part 5) for your standard curves; you will be plotting them (on the Y axis) vs the mg of protein (on the X axis). You do not need to plot lines on the residual vs mg graphs; just tell us whether the points are one of:

1. low on the ends and high in the middle;
2. high on the ends and low in the middle; or
3. neither of the above.

On Kalidagraph (which is on the computers in 214 and the PCs in 202):

1. Make sure the equation you are using is something like (upper/lower case doesn't matter) y = m1 + (m2*m0);. Do not use a Michaelis-Menton curve, which is discussed in the Kalidagraph handout!
2. You can get the residuals by going to Curve Fit, General, down to whichever one you are using for the equation of the line (it should have a check by it), selecting it, then clicking on the arrow under "View" for the column desired (absorbance or, for Coomassie (as well as the A₅₉₅ one), A₅₉₅/A₄₆₆), whereupon you get a menu and should select "Copy Residuals to Datasheet" or something like that (it's the last one on the menu for the version of Kalidagraph around here). Then on your datasheet (the one with the values for what you used to plot the standard curve) should appear a column labelled "Residuals". Plot these vs mg to get the graphs required for part 5.
3. For the standard error of the estimate, in order to do the other part of question 5 than the residual graphs, you can get error bars indicating the standard error by going to Plot, then down to "Show Error Bars" or something like that, then make sure that on what pops up you select "Standard Error" in the boxes - not percentage of value or whatever. The lengths of the bars should be the same for each value (on a particular graph). Look at the line through the points and compare it to the error bars - for the first and last points, are the points further away from the line than the length of the error bars, or not? If a point is further away from the line than the length of the error bars, then the point in question is outside the standard error; if not, it is inside. You do not need to then remove such points, replot, etcetera (as in, you don't need to do the removal you may have done for the first lab's statistical analysis), unless you would need to remove the points in question anyway for some reason.

Some people are getting very large errors (as in larger than the concentration) for some of their methods. It is possible that this is perfectly correct; look and see if the method in question gives a concentration rather different from your other values. Also look to see whether you may need to remove some points from the standard curve and/or from the absorbances for the unknown.

10/15/05:
Three things:

• Dr. Chase did what I am guessing to be a typo in the statistical analysis handout (from lecture earlier), namely stating that part 5 was done with the residuals vs the ml. Since this is the only place that says this and he was concentrating on part 4 in this handout, I believe that you should instead be - as stated above - plotting residuals, for the standard curves, vs mg of protein. I have emailed Dr. Chase to confirm this, and also Rob (Shortell, who will be grading the analysis) to let him know that some people may have done it differently than I state above (or the same as I state above, if I'm wrong!) and this should not be penalized if they're following either the handout or what I say above. (Feel free to include graphs of residuals for the unknowns if you've already done them - just be sure to label them so Rob can tell them apart from the other residuals graphs!)
• If you get huge errors for the average-the-concentrations method and much lower errors for the slope-vs-slope method, especially on ones other than Coomassie, this appears to be perfectly normal (and a reason not to use the average-the-concentrations method!).
• Make sure you aren't plotting (for Biuret and Lowry) the values of the blank vs water on your graph - plot instead (with 0 protein) the absorbance of the blank vs itself (which should be 0). (For Coomassie, you should be plotting the 0 protein values (including on the unknown plot(s)) with an absorbance above 0, namely that of no protein, just Coomassie reagents, vs water. For UV, the blank is water, so plotting the blank vs water is the same thing as plotting the blank vs the blank.)

10/17/05:
Well, Dr. Ward has come along and erased over half of the stuff I had up on the board in 206/207, despite the rather large "SAVE!!!" I had up. I'll be back tonight (I got less than 5 hours of sleep last night, and need to go home and take a nap) and will try to put said stuff back up again someplace - perhaps 202. Well, I'm back, and will be available after 9:30PM for consultation. Dr. Chase has dropped a sheet on my desk regarding the oddities we've been seeing in the results for the standard error calculations, with a revised version of the analysis - but one that will take rather more time to do. You will be given credit for any variety of error analysis that takes into account the error of the standard curve, so the old version will be given credit, don't worry.

BTW, I am seeing people doing the x = (y-b)/m calculations for each and every data point in the standard, due to following literally the material in Dr. Chase's handout. This section of said handout was meant as an explanation of what was happening, not as something you were supposed to be doing. The unknown is what you figure out this calculation for, and this should have been done last week (with the main protein lab report), for everything except the A₅₉₅ on the Coomassie.

10/19/05:
Due to tiredness with the first enzyme lab session (the last people left at ~7:30 PM...) and that Dr. Chase has made some changes to the methods involved, I haven't put back up the material on the statistical analysis. I do have some sheets with (most of) the material in question on them, and can make copies.

In regard to the carbohydrate labs, while I may be available this weekend and will be available next Monday late afternoon/evening, I am not a good person to help on them. (I'm a geneticist - the only carbohydrates I know much about are those involved in DNA and RNA!) I recommend starting with your general biochemistry textbook(s) (for information on which carbohydrates have what characteristic), then checking with Dr. VanEs and/or Dr. Chase. If consulted, in most cases I will simply be checking textbooks (and/or online) myself! I am thus worried that, should I be consulted and tell you something, it may be incorrect, and I don't want to mislead people (that I know more about this than I do).

10/20/05:
A clarification on the above regarding the carbohydrate labs: I can answer questions on graphing and on the curve-fitting (mutarotation analysis) that is due a week later. But questions on the identification of the carbohydrates or on the answers to the questions at the end of the lab should be asked of Dr. Chase and/or Dr. VanEs (whomever you can catch first, basically...), after you try to solve it yourselves using the information in your course textbook, online, etcetera. (We do have some additional textbooks in 119 that you can look at - but may not remove from the room without permission, and may not remove from the building in any event - if your general biochem textbook is lacking this year. (If it is lacking, I encourage informing Gavin and Dr. VanEs about it, BTW!))

10/21/05:
A few things on the carbohydrate lab:

• You do not need to do intensive statistical analysis (residuals, standard errors, and similar) on your data; you will be doing the mathematical analysis on the mutarotation data that Dr. Chase gave you, which is due a week later than the rest of the carbohydrate lab. So far as I know, all that you need to do for the graphing on your data is getting the best-fit line (don't force it through 0!), with equation and R-squared value.
• Remember that you need a 0,0 value on the standard curve graphs, except for the ferricyanide standard curve. The ferricyanide method is a disappearance method - the point you plot for 0 carbohydrate should be the highest value for the ferricyanide method.
• There are two seperate things to submit to turnitin.com on the carbohydrate lab (and remember that turning things in late to turnitin.com does count more points off!):
  1. the conclusions as to what carbohydrate you have and (more importantly) why you think this is your carbohydrate - within a lab group, saying the same carbohydrate is fine (indeed, preferable!), but you should write up your reasoning seperately
  2. the answers to the questions

10/28/05:
Two things:

• I'll be trying to put together and put up (here) some info on how to do the mutarotation analysis if people are using Sigmaplot (Dr. Chase covered it for Kalidagraph). See below, 10/30/05.
• I have a presentation for a seminar class on Monday, and will consequently not be available (in general - you can ask me to do things like signing labs as in on time or whatever) until after that. After said class, once I have time to collect myself, I will be available for assistance with the mutarotation analysis that afternoon and evening.

10/30/05:
As per the above, I am not available (in general); anyone bothering me unnecessarily may get something thrown at them! I do have a temper... For the mutarotation writeup for how to do it on SigmaPlot, see mutarotation.sigmaplot.txt.

10/31/05:
As it turns out, there are two different sets of numbers for the first-order decay curve, one for glucose, one for xylose (including some changes in the text - namely the 7.7 (for xylose) that one subtracts). My mutarotation setup for SigmaPlot was done with the sheet for xylose, which I had thought was the only one. Substitute numbers as necessary to make use of it for glucose, or just get a xylose sheet and do it with those numbers.

I will be available for helping with the lab as of roughly 3:40PM, and will probably be here most of the afternoon and evening.

11/2/05:
Some things on mutarotation (the assignment due this week from the sheets Dr. Chase handed out):

• For the equation for the fourth graph, among the starting values is one with a minus sign (m2 for Kalidagraph); the minus sign is unfortunately hard to see - but this value must be initialized with a negative value (the computer can't adjust it through 0, since it's multiplied times x (m0 for Kalidagraph)...). If you come along to a computer with Kalidagraph and the equation already in there, make sure it has the right starting values!
• Be sure you have the equation right! I've seen people with plus signs instead of multiplication (*) signs, for instance; again, be sure that the equation is the right one if you find it already on a computer.
• So far as I know, there is no facility in Kalidagraph to do the number-minus-something or ln-of-number calculations. You can:
  • Use SigmaPlot, which does have said facility;
  • Use Excel to do the calculations and import the results into Kalidagraph; or
  • Do the calculations by hand (using a calculator).

11/7/05:
Please refrain from smoking outside the side door closest to my office (and outside any other doors if you notice a window open), at least until they get the heating system fixed so that it isn't over 80 degrees in 118 (my office) even with 2 ACs on. Try the benches in front of the building. If I catch you smoking outside the side door when I have the window to 117 open, I will not be happy...

11/9/05:
Several things (some important for all students in the course!):

• I will not be available this weekend, due to having a presentation on Monday (for a graduate seminar course I'm taking - yes, TAs take courses too!). I had actually originally been scheduled to do the presentation a week from this Monday, but:
  • The student who was doing it next Monday was having major problems with that schedule due to being out of town most of this week; and
  • Probably more importantly from y'all's viewpoint, this means I will be (barring unexpected events) available for consultation on the weekend before Thanksgiving break on the enzyme lab.
• I advise starting ahead of time to work on the enzyme lab. Dr. Chase will be grading it (this means, among other things, that you should make sure that your graphs look different - different symbols, etcetera; he'll pick up on this (and care) when others would not; just make sure they don't look as if you just printed copies for your entire lab group!), and is the one to consult first if he's available, but I can do quite a bit of it (and am probably the best one to handle curve-fitting on the computers, provided I know exactly what Dr. Chase wants you to hand in - so it is most definitely best that I have time to check with him if there are any questions!), and Laura and Rob - especially taking mercy on the latter, please, since this is his first time! - can likewise help you with much of it (such as interpreting what is meant).
• Neither the lab nor the lecture for the lab will be happening the week of Thanksgiving break.
• If you manage to hand in the lab report before Thanksgiving, it's 5 points extra credit, and that will be multiplied effectively by 4 because the lab is a 4-week lab (it counts 400 out of the ~1600 points the final grade is out of). But turning it in on time (not late, if at all possible! late points are also multiplied by 4, effectively!), well-done, is going to help you more than turning it in before Thanksgiving, not well-done.
• For the extra credit, I would not advise spending time analyzing data that is, well, crappy. But if you have usable data, since you spent the time collecting it, you might as well get the points for it by analyzing it (well!).
• There are two entries on turnitin.com for the enzyme lab, one for the procedure, one for the questions. Please don't put them in the same file! Use two seperate files. And don't put the questions into the file for the questions - just the answers. People doing otherwise on the carbohydrate lab have given me a headache which does not incline me to be nice to the people in question... (Yes, we know, the numbers within a lab group will all be the same for the procedural writeup. But your sentences should be different enough - just as with the conclusions on the carbohydrate lab - that we can tell you wrote them seperately.)
• There are some people who haven't yet turned things in on the carbohydrate lab on turnitin.com - please do so immediately so no more late points come off! Sorry about not letting people know earlier, as I did for the protein lab, but I've been rather busy with one thing and another...

11/19/05:
I'll be available Sunday (11/20/05) afternoon (after 12:00 noon, literally) and Monday after Biochem 403 (as in 5:15pm or so) for assistance with enzyme labs. I may be available other times, such as right now (Saturday evening), Sunday evening, and Monday afternoon - email or (if the building is unlocked) drop by to check. Dr. Chase says that the extra credit points will count if you have it in anytime on Tuesday (11/22/05), including after midnight, and up till some time on Wednesday - as in, if it gets to him before he leaves on Wednesday, you get the points. (I am likely to be here all Wednesday and after that during the Thanksgiving break, since my nearest family is in Georgia, and may be available to help with enzyme labs but email first to check!)

BTW, if you are outside waiting for someone to let you in, as well as calling the various phone numbers (119, 206/207, and/or 202), if the window next to the door by my office (the one closest to Loree) is cracked, try yelling. (You can also try yelling to see if anyone in 214 - the small computer lab - hears you. If you hear someone that you know/trust yelling to get in, please do so! Also, if you hear the phone ring in 206/207 or 202, answer it, and if it's someone you know to be adequately trustworthy needing to be let in, please let them in!) Also try tapping on my window with a stick or something, especially if the light is on in my room.

Regarding question 5 and similar questions involving Michaelis-Menton plots of substrate vs rate:

• I generally find it best to produce a linear-form graph first, then use it to get estimates of the Vmax and Km before trying to fit a nonlinear curve. The most accurate one is probably the Woolf (or, to be precise, Hanes-Woolf) plot: see Hanes-Woolf.new.gif, originally from http://opbs.okstate.edu/5753/Kinetics/hw_plot.html but with modifications. "[S]" is, of course, the substrate concentration (e.g., the D-Alanine concentration) and v (in "[S]/v") is the rate (e.g., (umoles pyruvate)/(minute*(ml stock enzyme)))
• Once you have estimates for the Vm and Kmax, you can use these and SigmaPlot or Kalidagraph to get a nonlinear curve fit using the Michaelis-Menton equation (v = ((Vmax*[S])/(Km + [S]))) with the estimates used as initial values for Vmax and Km. This is rather easier to show than describe, although you can try looking in Dr. Chase's Kalidagraph handout for how to do it, so I am not going to attempt to describe the process (at least for now) - if you are uncertain how to do this, you will need to get me (or - for SigmaPlot - Dr. Kahn, or - for Kalidagraph - Dr. Chase) to show you how the first time.
• For the Hanes-Woolf (or Lineweaver-Burke, useful for figuring out inhibition) plot, do not plot a 0,0 point (for the Hanes-Woolf plot, for instance, this would be 0 on the X axis for 0 substrate - fine - but then 0/0 on the Y axis for 0 substrate and 0 rate, which is undefined). But do plot a 0,0 point (0 substrate, 0 rate) for the full Michaelis-Menton (nonlinear) plot.

11/21/05:
One thing to keep in mind when calculating substrate (D-alanine) concentration for Michaelis-Menton and related graphs (e.g., Hanes-Woolf) is that this is the concentration in the reaction. 0.06 M DL-Alanine = 0.03 M D-Alanine; 0.03 * an amount in mL gives you mM (millimoles), but the (pyruvate assay) reaction is occuring in 1 mL volume, so you divide mM by 1 mL and that gives you back Molar. You can make this number more convenient by multiplying by 1000, provided that you then label [D-alanine] as something like [D-alanine] (mM), since [D-alanine] would normally mean molarity of D-alanine, not milli-molarity of D-alanine.

I am heading out now - it's about 10:20PM - and won't be back tonight, unless I wake up in the morning and can't get back to sleep, which has been happening recently. Hope nobody was banking on my being in very late tonight - but I would like to go get some food and sleep, partially in order to be available later Tuesday night!

11/22/05:
Note for figuring out the molar extinction coefficient for experiment 2 that you need to allow for any dilution used to get the spectrum. Divide the protein concentration (in mg/mL == g/L) by the dilution factor before converting it into molarity (moles/liter). This is something that I've been forgetting at times - sorry! - partially because I saw a bunch of people who were using undiluted enzyme for experiment 2.

In regard to the 5 points extra credit that you get for handing the lab in before Thanksgiving, yes, it's effectively times 4 due to the lab being worth 400 points - so it's 20 points out of 400. There has been some confusion on this...

Another thing on extra credit: I'm afraid that experiment 7 is, by default, only 5 points, not 10 points; I had been thinking it was 10 points like the rest - sorry!

Note that if you did experiment 8 as well as experiment 5, you may want to use a Lineweaver-Burke plot to get the initial values for a Michaelis-Menton plot for both experiment 5 and experiment 8, since Dr. Chase wants you to graph using Lineweaver-Burke (and Michaelis-Menton, possibly - this is unclear; you definitely need to do a Michaelis-Menton plot and curve fit for experiment 5) for experiment 8 and include a line for experiment 5 on that graph. This would be instead of doing a Hanes-Woolf plot.

Dr. Chase has stated in regard to the inconsistencies in the section on "The Report" (the below is a quote from an email from him, replying to one from me):

Allen Smith wrote:
P.S. BTW, in the section "The Report", you ask for assay and protein concentration data in tables and graphs in the account of purification. Then you say to give a partial description of the experiment (assay?) procedure, then graphs, then explain the data. Is all this interleaved in with the account of purification, including descriptions of how to do the assays, or is it after the account of purification? And after that come the experiments (as opposed to the assays)?

Dr. Chase wrote:
On paper I could take it either way; indeed, the purification may be easier to follow if the data are interweaved with the account. But they should save a data-free Account of Purification to send to turnitin.com.

In regard to whether you are required to graph absorbances vs DEAE-Sepharose fractions, no, you aren't, since it's not in the lab manual. But Dr. Chase would appreciate it if you did so if there was any lack of clarity in which fractions it was best to take, unless that would mean you would turn the lab in much later (as in late enough to not get extra credit points or to get late points taken off).

Dr. Chase will be here until sometime in the middle/late afternoon tomorrow (11/23/05); he is not likely to be here after 5 PM.

11/23/05:
Reminder: You also need to submit the procedural writeup and the answers to the questions, in separate files, to turnitin.com. You should remove everything but the text of the procedure and the text of the answers to the questions (do not include the questions themselves) before you upload these files. (E.g., do not include graphs or tables!)

People who live a significant distance away can try faxing lab reports to the departmental fax (see http://aesop.rutgers.edu/~dbm/ for the number; I don't want to give it here in case it changes, which has been known to happen...), which we will try to check regularly today. You should probably also email to Dr. Chase (and possibly myself) that you are doing so, possibly including a copy of the lab as an attachment in the email to Dr. Chase only (not to me, I don't do attachments!). But faxing is (usually) much more reliable than email attachments, especially for stuff from Kalidagraph, Sigmaplot, and to a lesser degree Excel.

I should be available to answer questions until Dr. Chase leaves, except that between 2:30 and roughly 3:30 I will be meeting with Dr. Kahn (my dissertation research advisor) and should not be disturbed. After Dr. Chase leaves, I would rather like to be able to take a break (I think I'm coming down with something - post-nasal drip is starting up...). I should be available (with more time between question and response) via email, however. I will endeavour to be available in person more time after today, and will try to post up more information as to when I will be specifically available - you can also inquire of me via email as to any other times I might be available in person.

11/27/05:
I am seeing a problem with a few people's graphs that were done on Excel:

• They used a line plot instead of a scatter (XY) plot, with the bottom axis being treated as categorical data instead of as numbers; and/or
• The values for the amounts of enzyme/pyruvate/whatever were being treated as plain text, not as numbers (to fix, select the cells in question, go to Format, to Cells, and tell it that they aren't in "General" format, they're in "Numeric" format), again resulting in their being treated as categorical data.

12/3/05:
I'll be here most of this weekend and available (as I have been most of the past week) for enzyme lab work - email if you need to make sure I'm here and available at a particular time, keeping in mind that I'm currently up until 4 AM or later most nights/mornings... (BTW, a request for suggestions: What's the best way to tell people in the course catalog or whatever about exactly how much time this course can take? Including both indicating that labs can last over the technical periods (which should probably be extended) and the amount of time needed for writeups for the enzyme lab and similar? Only give suggestions that various bureaucrats would be willing to put in, of course! That's what makes me ask for suggestions... I am not good at being diplomatic, at least in writing.) Also feel free to email questions about the lab report (or lab in general); I much prefer email to phone calls, BTW! However, keep in mind that I generally can't receive attachments.

People who have turned in their lab reports on paper: Do remember that you also need to submit, into their respective seperate categories, your account of purification (minus tables, graphs, and calculations) - as "Enzyme Lab Procedure" - and the answers to the questions (again, without any tables, graphs, or calculations - just the text), to turnitin.com. After I check with Dr. Chase on Monday regarding who has turned it in on paper, I will try to send out reminders, but no guarantees; I am absent-minded!

My schedule on Sunday should have me available between 4 PM and 7 PM at the minimum; email for whether I will be available at other times - my current sleep cycle is being thoroughly erratic (I got to sleep this morning at 4 or 5 AM, woke up at 9 AM (yuck!), got sleepy around 2 PM but didn't take a nap, did take a nap from 7 PM until now (11:50 PM), etcetera...)

12/4/05:
I should be available between now (4 PM) and 11 PM. I may be available after that - I will likely be here most of the time until around 3 or 4 AM Monday morning - but may go out to get something to eat.

Well, it's now 2 AM. I will be here until 2:30 or 3 AM (the latter only if someone emails me to tell me you're coming with a lab report to turn in - I'm getting sleepy, and would like to be safe driving home!), then I'm out of here. I will, however, be available most of the afternoon and evening on Monday.

12/6/05:
Well, I had thought I'd be around this evening to start the analysis, but I am feeling rather exhausted, and nobody seems to be around. If you were meaning to work on the gel picture analysis this evening and wanted my help, my apologies and let me know... but I'm needing some sleep (I was up at 9 AM this morning to do grading, after a week in which I was going to bed at ~4 AM...)! I should be available most of the other afternoons/evenings/nights this week except Friday (and maybe Thursday - don't know on that one yet).

Well, I'm actually back here at ~9 PM, after a short nap. I should be available until 1 AM (at least), and am planning to be then available again after 1 PM or so Wednesday.

I will post up the isoelectric gel pictures on this website when I get data on which lanes are which and on what the standards are. All the gel pictures for the Tuesday lab, including for the isoelectric gels Gavin did, are also on the computers in 214 under "Sec01 Pics" or something like that (in the "My Documents" directory). Demo copies of TotalLab, as well as the full-scale version on the picture-taking (Fotodyne) computer in the lab, are on the computers in 214 also; the demo version should hopefully be sufficient. There is a section in the lab manual on how to do the TotalLab analysis; I can also be consulted (in person or via email), as can Dr. Chase. If I run into any problems that you need to know about, or anyone tells me about such problems, I will post this information here, hopefully along with solutions.

I am grading the electrophoresis and isoelectric focusing labs. I greatly prefer everything but calculations being typed, but do not require it (although it will help subjective credit for people in my section!). I don't want a full writeup of procedure; just tell me all amounts, measurements, deviations from the procedure in the lab manual, problems noticed, etcetera.

12/7/05:
I am here, and beginning to wake up (I was up until around 4 AM). Peter Anderson is working on getting the TotalLab software working in the PCs in 214. I will do a scan-in of the isoelectric focusing gels from Wednesday sometime today, and post up those gels and the Tuesday ones also, perhaps with a bit of fiddling (mostly to put on info on the lanes); Gavin should be sending me the info on the standards today. Once Peter Anderson gets the TotalLab software working in 214, I'll cart over the Wednesday images to the PCs in 214 (and maybe the Friday ones from last week, then I'll put on the Friday ones from this week on Friday) and Dr. Chase will send out an email to the class letting everyone know that the computers in 214 are able to analyze the data.

Here are the isoelectric gel pictures for Tuesday, in multiple versions and file formats. Putting together the different exposures worked OK for the 3-9 pH range one but somewhat weirdly for the 4-6.5 pH range one.

• 3-9 pH range:
  • Exposures combined:
    • iso.3-9.anode.tab.tif
    • iso.3-9.anode.tab.png
    • iso.3-9.anode.tab.jpg
  • Medium exposure:
    • iso.3-9.med.anode.tab.tif
    • iso.3-9.med.anode.tab.png
    • iso.3-9.med.anode.tab.jpg
  • High exposure:
    • iso.3-9.high.anode.tab.tif
    • iso.3-9.high.anode.tab.png
  • Low exposure:
    • iso.3-9.low.anode.tab.tif
    • iso.3-9.low.anode.tab.png
• 4-6.5 pH range:
  • Exposures combined:
    • iso.4-6.5.anode.tab.tif
    • iso.4-6.5.anode.tab.png
    • iso.4-6.5.anode.tab.jpg
  • Medium exposure:
    • iso.4-6.5.med.anode.tab.tif
    • iso.4-6.5.med.anode.tab.png
    • iso.4-6.5.med.anode.tab.jpg
  • High exposure:
    • iso.4-6.5.high.anode.tab.tif
    • iso.4-6.5.high.anode.tab.png
  • Low exposure:
    • iso.4-6.5.low.anode.tab.tif
    • iso.4-6.5.low.anode.tab.png

• 3-9 pH range:
  • Exposures combined:
    • iso.wed.3-9.anode.tab.tif
    • iso.wed.3-9.anode.tab.png
    • iso.wed.3-9.anode.tab.jpg
  • Medium exposure:
    • iso.wed.3-9.med.anode.tab.tif
    • iso.wed.3-9.med.anode.tab.png
    • iso.wed.3-9.med.anode.tab.jpg
  • High exposure:
    • iso.wed.3-9.high.anode.tab.tif
    • iso.wed.3-9.high.anode.tab.png
  • Low exposure:
    • iso.wed.3-9.low.anode.tab.tif
    • iso.wed.3-9.low.anode.tab.png
• 4-6.5 pH range:
  • Exposures combined:
    • iso.wed.4-6.5.anode.tab.tif
    • iso.wed.4-6.5.anode.tab.png
    • iso.wed.4-6.5.anode.tab.jpg
  • Medium exposure:
    • iso.wed.4-6.5.med.anode.tab.tif
    • iso.wed.4-6.5.med.anode.tab.png
    • iso.wed.4-6.5.med.anode.tab.jpg
  • High exposure:
    • iso.wed.4-6.5.high.anode.tab.tif
    • iso.wed.4-6.5.high.anode.tab.png
  • Low exposure:
    • iso.wed.4-6.5.low.anode.tab.tif
    • iso.wed.4-6.5.low.anode.tab.png

• 9.6 This will not actually show up as a usable band on the IEF gels, because they are from 3-9 or from 4-6.5
• 8.2
• 8.0
• 7.8
• 7.5
• 7.1
• 7.0 Note that standards above 6.5 will not show up as a usable band on the IEF 4-6.5 gels
• 6.5
• 6.0
• 5.1
• 4.75
• 4.65
• 4.45

1. Dan (2.1 mg/ml)
2. Josh (1.43 mg/ml)
3. Ying (4.745 mg/ml)
4. Standard - the lane with all the bands in it
5. Jamie (1.15 mg/ml)
6. Gonzalo (1.34 mg/ml)
7. Xi (4.0 mg/ml)
8. Gagandeep (sp?) G, M (x/4 mg/ml - check Dr. Chase's concentration calculation)

1. Mike (3.97 mg/ml)
2. Max (3.56 mg/ml)
3. Jon (1.17 mg/ml)
4. Dipen (5.035 mg/ml)
5. Standard - the lane with all the bands in it
6. Ron (1.335 mg/ml)
7. Ambrish/Rochi (2.185 mg/ml)
8. Katie (2.15 mg/ml)

One of the PCs in 214 now has the TotalLab software running on it, but this is currently instead of the 207 FotoDyne PC having it - it kept crashing on the FotoDyne computer, so Peter Anderson has moved it over. Hopefully it'll crash less on a more-updated version of Micro$loth Windoze... Peter Anderson is still working on getting the other machines working with a demo version - the software company is being very sluggish.

12/8/05:
Well, with a more-updated version of Windoze, TotalLab seems to crash almost as often, but at least it doesn't take the entire rest of the OS along with it quite as much. Save as frequently as possible, including what you've exported to Excel! Peter Anderson is still working on getting the other machines going with a demo version, so far as I know. In regard to what you need to analyze using TotalLab, you don't need to analyze the samples from the other group (unless yours are unusable, and you state that you're using their data - with their permission!), analyzing the activity-stained native gel with it is optional (possible extra credit), and you can skip analyzing your native blots with it if it's impossible to distingish bands on the picture. I'll be available Saturday, Sunday, Monday, and Tuesday to work on the lab - not sure as to exact hours as yet, except that I'm currently not awake until the afternoon at the earliest (although I have a 12:35 class on Monday that I'll need to get up for... groan!). I will not be available on Friday - I first have an appointment with Dr. Kahn to get ready for, then I will be having said appointment, and then I'll be taking a break and eventually going to a party... I suggest y'all also take a break!

In regard to whether you can just do an "automatic" analysis on TotalLab, this depends on whether it works for your gel and blot pictures or not. If it detects all the bands (that you need to analyze, which normally doesn't include those from the other group) that you can see in your pictures (and/or can see on your gel/blot themselves if you still have them, which you should for the blot), then it's fine to use the automatic results. Otherwise, you need to use the manual mode.

12/9/05:
TotalLab is now up and running on all computers in 214; a thank-you for the work involved in this goes to Peter Anderson (so keep 214 clean!). I am not available for the rest of the day, except that I may (if I notice and am feeling like it) answer emails. I will probably not be up until 2 PM on Saturday and Sunday, and possibly not until 3 PM.

12/10/05:
I'm here and (groggily) awake. Several things on the electrophoresis and isoelectric focusing labs:

• I may not use the same point scheme that Dr. Chase has in the lab manual. It is likely to be approximately right, but I may well alter it depending on what I see in people's reports (I am likely to remove any points for analyzing the native blot and make that extra credit or something, for instance).
• Rfs (aka Rms) should be approximately equal between the gel and blot (more so than the cm or pixel (from TotalLab) measurements). These are gotten by (distance from top to band)/(distance from top to dye front).
• The weights for the prestained (different colors, and visible even before staining with Coomassie or on the antibody-stained blot; aka stained) standards are on Emilia's door (marked with an X); do not use the ones from the lab manual - they may not have been updated (or they may have; I haven't checked, and the picture on Emilia's door is useful in any case).
• The weights for the non-prestained (only one color (or two, if you used the Coomassie-stained gel to do the antibody-stained blot), and visible only after staining with Coomassie or on the antibody-stained blot; aka unstained) standards are on page 16 of the lab manual.
• You should graph both the prestained and the non-prestained standards (log(molecular weight (MW)) vs Rf) and see if they line up along the same line. If so, use both of them. If not, give both graphs, and use the non-prestained standards for determining molecular weight - except that, if the prestained standards show up on your blot and the non-prestained standards do not, and the Rfs for the blot sample lanes are different from those on the gel, you will need to use the prestained standards graph equation to get the molecular weights for the blot bands.
• To get molecular weights, you will take the equation of your line for the graph of log(MW) (on the Y axis) vs Rf (on the X axis) for the standards, plug in the Rfs for your bands on the other lanes into the equation as X, get out the log(MW) as Y, and then take the antilog to get the molecular weight (MW). (Be sure that, if you did log-base-10, you then do 10^Y to reverse it, and not e^Y, or vice-versa! A good idea to doublecheck for this, and for other errors, is to give the molecular weights that your equation would give for the standards as well as the known weights for the standards in your table, and make sure that they look to be about the same (within 1000 or so, or within 100 or so for standards with sizes below 10,000).)
• I will put up pictures of the Friday lab IEF gels as soon as someone tells me either where the gels are or where the picture files are... plus info on which group is in which lane!
• Keep in mind that the TotalLab software Rfs are to the bottom of the picture/defined lane, which may not be to the dye front! The dye front often shows up as a band of its own. You will need, in such cases, to do the division yourself instead of just using the Rfs that TotalLab produces.(If the dye front does not show up as a band of its own, and you can see it on the picture, put it in manually as an additional band!) Also keep in mind that your picture may wind up being inverted (top of gel at the bottom) or sideways; you can adjust for this in TotalLab by editing the image (one of the icons on the top line, I believe the furthest to the right) and saving a new image with the appropriate corrections made. You can also clip off unneccessay parts of the image in the editing mode.
• With regard to manual measurements on gels/blots that you also are going over using TotalLab, you need to at least measure the distances to all the standard bands (especially if they are colored - the prestained standards - which do not show up as well on the greyscale photographs that TotalLab is using) and to the major bands in your sample lanes (as well as to the dye front in all cases). Calculate Rfs using these as well as TotalLab, partially to serve as a check on whether the values you get from TotalLab are being interpreted properly. If you are only required to do either the manual analysis or the TotalLab analysis, and are doing both for extra credit, you do need to do both fully - all visible bands - for said extra credit.
• With regard to the isoelectric focusing gels, you only have to analyze one of them - either the 3-9 or the 4-6.5 one, whichever you prefer. Doing both is extra credit.
• With regard to the IEF, I am looking at it and am now not sure that the end with the tab has the lowest pHes; the standards look to be the other way around. I am attempting to clarify this with Gavin and Dr. Chase.
• Again for the IEF, you start with the top of the lane being at one of the two lines (lighter area between two darker areas) that goes across the gel (where the electrode was) and end at the other of the two lines.

• SDS coomassie-stained gel, required:
  • TotalLab
  • Manual (at least the most significant bands, including all on the standard lane(s); ideally (possible extra credit) all)
• SDS blot, required:
  • TotalLab
  • Manual (at least the most significant bands, including all on the standard lane(s); ideally (possible extra credit) all)
• Native (activity-stained) gel:
  • Required: Either manual or TotalLab
  • Extra Credit: Both
• Native blot:
  • Required if usable: Either manual or TotalLab
  • Extra Credit: Both

For getting extra credit for doing manual in addition to TotalLab, calculate all Rfs, plot the Rfs of the standards against their log(MW), plot a best-fit (least-squares) line through these, compare this to your TotalLab results and note any differences (and possibly combine with your TotalLab standard graph), then take each of the Rfs you get manually and determine the molecular weight, comparing to your TotalLab results. For doing this for the SDS gel, I am currently thinking of 5 points minimum extra credit, with more possible if not many people do this. Ditto for the SDS blot. I am not sure yet for the native gel and blot.

Despite what it currently says in the lab manual for some reason, you do not need to use a log scale on the Y-axis for a log(MW) (on the Y-axis) vs Rm/Rf plot. Indeed, I am likely to find your doing such rather confusing! I will need the numbers corresponding to log(MW), not MW, to see if your equation is reasonable. A scale consisting of "1" at the bottom and "10" at the top, for instance, is not desirable; instead, show something like 4 at the bottom and 5 at the top. The only time I should be seeing a log scale on your standards plot is if you manually draw the standards plot by hand on semilog graph paper (with the units on the Y axis being molecular weight, not log molecular weight), then cross-reference vs a manually-drawn line to get molecular weights from Rms/Rfs. (This is specifically permitted as an alternative to plotting log(MW) vs Rm/Rf and getting the equation of the line then using that to find your log(MW), then doing an antilog.)

With regards to the amounts that you put on the gels:

• Show the amount, in ul, that went into each lane
• State how you calculated or otherwise determined the above, for everything except the standards
• For the SDS gel, show what the ug that said lane has in it, from your mg/ml values from the enzyme lab (if Dr. Chase corrected your protein concentrations, use his corrected mg/ml values), as in multiply the ul used by the ug/ul (which is the same thing as mg/ml) to get ug.
• For the native gel, show what the units of activity are that said lane has in it, from your units/ml values from the enzyme lab (if Dr. Chase corrected your activity per ml, use his corrected values), as in divide the ul used by 1000 to get ml, then multiply by the units/ml to get units.

Anytime you are plotting a best-fit (aka least-squares) line, I want to see the equation of the line and the R-squared value for the line. (On Kalidagraph, getting the R-squared instead of the R is done via the "Curve Fit Options".)

Note that you do not put a 0,0 point on the graphs of the standards in this lab report (including both electrophoresis and isoelectric focusing). You will get very bad results if you do!

You may need to take out points - for instance, if you use both prestained and non-prestained standards to get a graph of log(MW) vs Rf/Rm, you may need to take out a couple of the prestained standard points to get a good line. If so, show me both the graph before removing points and the graph after removing points, with equations and R-squared values for both, then use the better of the two graphs.

I do not make any distinction between Rf and Rm, BTW. Just show me how you calculated it and what the value you used for your dye front was.

Note that you should not be plotting anything except the standards, unless you are determining the molecular weight manually via a semilog plot. The instruction in the lab manual to plot the D-amino-acid-oxidase band is referring to if you are using a semilog plot.

12/11/05:
Three things on the questions:

• Question 2 is about why can we use a buffer without SDS for the lower reservoir buffer in the SDS gel (as well as the native gel). "Why don't we need SDS in the lower reservoir buffer?", in other words.
• The second part of question 5 is on why do we use two antibodies, instead of just attaching a visualization enzyme to our anti-D-amino-acid-oxidase antibody.
• Most importantly, do the questions seperately!

I am informed that the Friday lab didn't do IEF; just use either the Tuesday or the Wednesday pictures, telling me which sample lane you chose to analyze.

As it turns out, you are not required in the lab manual to calculate MWs for anything other than the band that you think is your D-amino-acid-oxidase band (which should be the strongest band on the final enzyme lane, but may not be the strongest band on your dialyzed precipitate ("after dialysis") lane). If you do calculate it for all bands, that will be extra credit.

I have made a couple of notes on the IEF standards, including below. Note that if you analyze the IEF pictures on TotalLab, you may need to go in and put in "grimaces" on some of the lanes due to the bands being s-shaped or otherwise curved; see the Tuesday 3-9 gel's standards for an example. I will try to put up labeled versions of the IEF gels before long.

12/12/05:
In regard to what should be in the SDS gel and blot tables, the lab manual currently mentions the prestained standards only for the gel table. These go on the gel table only if you have TotalLab and/or manual measurements of distances, and thus Rfs/Rms, that likewise go on the gel table (because they were determined from the gel). They go on the blot table, including information on the colors and molecular weights (from Emilia's door), if your prestained standards show up on the blot. (If you have data from the prestained standards for both the gel and the blot, then the color and other information goes on both tables.) Note that combining the blot and gel tables is also fine, as long as you are clear on what data comes from what source (blot/gel). You can put TotalLab and manual measurements on the same table or in two different tables, BTW; again, as long as they are clearly labeled, either is fine.

Note that the information in the lab manual about loading in a preferences file for TotalLab is not currently accurate, due to the multiple computers that TotalLab is now loaded on - just skip loading in the preference file; all the important options selected should be mentioned in the lab manual already. Also, the file location that it says in the lab manual is incorrect now - look in "My Documents" or "Student's Documents" or whatever, under a directory named something like Sec01 Pics (or Sec02 Pics, or Sec03 Pics, as appropriate for your lab section).

For the IEF standards: The tab (wedge-shaped thing) is next to the cathode, not the anode; the lowest pIs (pHes) are thus at the end away from it. The double/triple bands at the end away from the tab are the pI 4.45/4.65/4.75 standards (if it looks like 2 bands instead of 3, then two of them are merged together).

With regard to the final exam, I recommend going to Dr. Chase's review session tomorrow morning, as well as going over old exams and lab manuals. I may put together a question on making up a table like I did for the pyruvate assay (as in what's on the board), and/or on interpreting such a table. Note that Dr. Chase has updated what is up on his website - more of the old exams now have (annotated!) answers.

Contrary to some rumors, I will be accepting lab reports after tomorrow (with late points taken off unless there's a proper excuse, such as illness, of course). We will not be accepting lab reports turned in after the final for the course, however (as in the morning (groan!) of the 19th), unless you're in the hospital or similar. Note also that if you turn in the lab report sufficiently late (anything after Wednesday, definitely), I may not have time to grade it before the exam, in which case you won't get it back at the exam (I see no reason that I shouldn't be able to have those that are turned in on time back at the exam), and you probably will get a T grade when Dr. Chase turns the incomplete grades in.

In regard to the first part of question 5, what the question is asking about is why we don't just use the antibodies on the gel - why do we transfer the proteins to the blot and then use the antibodies on the blot?

In regard to IEF, you can do it on either TotalLab or manually. Doing both, if done right, will be extra credit.

12/13/05:
In regard to the discussion of purity (the paragraph on which in the lab manual has some text missing), the primary thing to do is to get the following ratios:

• (Band percentage of D-amino-acid-oxidase band in final)/ (Band percentage of D-amino-acid-oxidase band in dialyzed precipitate (after-dialysis))
• (Specific activity of final enzyme (from your enzyme lab, as corrected by Dr. Chase if applicable))/ (Specific activity of after-dialysis (dialyzed precipitate) enzyme)

I've put up some additional copies of some of the IEF gels, in JPEG (.jpg) format, after some people were having problems with the other formats. Sorry that I haven't had time to create and put up labelled versions, but I've been rather busy...

In order to determine which band is your D-amino-acid oxidase band on the SDS gels, look for which band in the final enzyme lane has the largest band percentage. If its molecular weight is around 39,000 (within 5,000 or so), it's almost certainly D-amino-acid oxidase. Then for the dialyzed enzyme lane, look for the band with the closest Rf. That should be your D-amino-acid-oxidase band in that lane.

BTW, when using either a flash drive or a floppy, be sure to close all programs that are using a file on the drive/floppy before you remove it! Doing otherwise can mess up flash drives and floppies on PCs, according to Peter Anderson.

12/14/05:
The final exam is in Ruth Adams 001, Monday, Dec 19th, starting at 8AM.

I am here and available for questions today until quite late (well after midnight - I was up until after 5AM this morning). I think that I will be able to grade labs I get today (or earlier, of course) by the final, if I am not disturbed after today (except for people to hand in their labs late, of course, although they can also be given to Dr. Chase) - I will be starting on the grading Thursday (tomorrow), plus doing research work for a meeting with my advisor (Dr. Kahn) on Friday, plus creating a question for the final. Labs turned in after today will almost certainly not be graded by the final. In other words, as of tomorrow whenever I come in, don't ask me questions on the lab in person or on the phone, and don't expect to necessarily get a response via email (much less get a timely response! Ask Dr. Chase or someone else (e.g., one of the other TAs) before you try emailing me as of tomorrow...). However, you can come in and ask questions (or inquire via email) and/or turn the lab in to me until I go home sometime this morning - it is currently just after midnight.

With regard to the purity determination, you only really need to use the band percentages from your SDS gel, not the SDS blot or the native gel. The SDS gel band percentages are affected not only by the amount of protein in the band, but by how well the antibody bound to the protein in question.

I have concluded that making people do manual measurements of both the SDS gel and the SDS blot, even limited manual measurements, was a bit silly - the purpose of making sure that the software wasn't messing up is satisfied by doing only one or the other. I will therefore be just requiring doing one or the other partially manually, with the other, if done, being extra credit.

12/17/05:
I should have the labs graded by the final. If I manage to get them done before then, which I doubt, I will post that information up on this webpage. (I really need to get through all of the labs that will be graded by the final before I can give grades for any of them, much less give them back, from past experience. I have attempted to reduce the likelihood of needing to revise my grading scheme after grading some of the labs, which has almost always happened in the past, by going through about 17 of the labs last night, in detail but without really grading them; I don't know how well this will work, however.) I do not advise inquiring about your lab report before then; grading does not put me in a good mood...

I can let people into the building and receive labs, but my ability to give any further assistance on lab reports will be extremely (time-)limited. Make sure (via email/phone) that I am here and awake and can let you in before coming over, please, if you need to me to let you in to work on the lab report and/or to receive a lab report!

I have created a question for the final related to how to do a procedural table such as the one I constructed for the pyruvate assay. The question should not be difficult if you understand the material instead of trying to memorize the table I put up.

12/18/05:
The departmental website is down; see:

• http://www.google.com/search?q=cache:c335fl19ln0J:aesop.rutgers.edu/~dbm/ex2000ans.html
• http://www.google.com/search?q=cache:Ykw_iYsU28EJ:aesop.rutgers.edu/~dbm/ex1998ann.html
• http://www.google.com/search?q=cache:BjItd8yHV88J:aesop.rutgers.edu/~dbm/ex1997a%2520master.html
• http://www.google.com/search?q=cache:xgLuCFFZLLEJ:aesop.rutgers.edu/~dbm/ex1996a.html
• http://www.google.com/search?q=cache:W62mWBs7D0UJ:aesop.rutgers.edu/~dbm/ex1994aans.html

The departmental website appears to be back up; see http://aesop.rutgers.edu/~dbm/tedchase.html!

I may be doing something on a purification table as well as the procedural table question, BTW. Most of the 10 points of my question(s) will still be on the procedural table, however. (The pyruvate assay table is an example of a "procedural table", and is the one I am deriving the procedural table question from - with changes!)

12/20/05:
All the electrophoresis labs that I got prior to or at the exam are graded, and with Dr. Chase (he'll be in this afternoon); the rest will wait. The grade range on them was quite wide (78 to 186) but the mean and median were quite high (mean 125, median 121). Good luck on exams, and have a good holiday!

1/16/06:
Welcome back! Lecture will be happening tomorrow (1/17/06) at which time you'll be needing to organize into lab groups and take a look at the lipid vs carotenoid manuals so as to decide which to do and start thinking about what sample to use. One group on Tuesday will be doing lipids on some spirulina algae that I picked up about a year ago (or on a new sample, if it's missing from the freezer). (Yes, I'm requiring that one group use this as a source material!) Note that there are safety concerns with regard to both the lipid and carotenoid labs, and that I personally have problems with chloroform and diethyl ether (both of which give me a headache and make me bad-tempered and have difficulty thinking, so do your best not to expose me to them - keep things in the hood as much as possible!). I will be giving a (safety) quiz next week.

Neither Tuesday (1/17/06) or Wednesday (1/18/06) labs will be happening this week. One reason for this is that copies of the lab manual are not available yet; the xerox machine keeps jamming, and finally did so in a way that will require a service call to fix.

If you are not able to arrange for a group in/after the lecture, come to the appropriate lab section. At the lecture will be handed out copies of the lipid and carotenoid lab manuals; also either come to the appropriate lab section or email Dr. Chase or myself for any needed consultation on which to do, what source material to use, etcetera.

2/24/06:
The HPLC files are (growl!) again transferred to the computer next to the window in 214. They're in the Shared Documents directory.

I will be available to only a limited degree this weekend for helping with lab reports - I have quite a bit of research work that my advisor, Dr. Kahn, is pressing me to get done, including preparation for more than one presentation. I am also not the best person to ask on anything to do with carotenoids other than getting the computer to work properly. On lipids, I can assist with interpretation of the GC results and can start you off with how to use http://lipidbank.jp.

I will put up on here the picture of the test gel that I ran either sometime this weekend or on Monday - remind me Monday if it isn't up by then, please.

2/27/06:
The test gel for the Tuesday section, with formats as per the filenames:

• test.gel.tif
• test.gel.png
• test.gel.jpeg
• test.gel.gif

1. NEV
2. SV
3. MKD
4. SYS
5. RJF
6. JYX
7. CJF (B)

Dr. Chase has given me information on what, for the carotenoid and lipid labs, should be sent to turnitin.com - I will try to set this up this evening (and if I am delayed by problems with turnitin.com or whatever, it won't be held against you, of course!):

• Carotenoid lab: Discussion
• Lipid lab:
  • Account of extraction
  • Conclusions from the plates
  • Account of preparation of methyl esters

Dr. Kahn appears to have forgotten about the meeting we were supposed to have this afternoon, BTW... sigh! Dr. Kahn turns out to have a stomach bug - I'm guessing the one that I fortunately fought off and I know at least one student in the Tuesday section had a lot of problems with; hopefully, he'll be better soon!

In terms of getting the names of the fatty acids that showed up on GC:

• Sometimes it gives more than one possibility. Some of these, with "ANTEISO", "ISO", "3OH", or "2OH", are less likely for most sources (the GC is programmed to be able to identify bacterial lipids, which tend to include weird ones). (You might want to take a look at www.lipidlibrary.co.uk on these.)
• On lipidbank.jp, the position in the search form is counting from the acid end of the fatty acid (the one with the O), while the "w" (actually supposed to be an omega symbol) form (w7c, for instance) is counting from the opposite end. So, if you were wanting to look up what 16:1 w7c was, you would put in 16 carbon atoms, 1 double bond, and the position of the double bond being on the 9th carbon (16-7), not the 7th.
• There may be multiple possibilities for a given description (number of carbons, number of double bonds, position of double bonds). I would look first at the ones that have a common name (in bold on lipidbank.jp) and then at any notes of what they are found in. Ones with a common name are, well, likely to be more common!
• If the GC says "Column Overload", meaning that there was so much fatty acid coming off at a given time that it had problems figuring out the peak (the retention time may be somewhat uncertain), try looking at any other GC results that you have for other fractions of the same source (or, if need be, those of other people in the course who were using a similar source) for something with roughly the same retention time that you do have a name for. (If the "ECL" column gives close to a whole number, then it may be a saturated fatty acid with that number of carbon atoms.)
• If there isn't a common name, try to give the chemical name - an organic chemistry textbook may help here.

I've created the Tuesday section on turnitin.com; the password is the same as last semester (I'll probably send out an email with it tomorrow), but the class number for the Tuesday section is 1479766. I will create a Wednesday section once I get some info from Laura. Note that the turnitin.com due dates for the Lipid and Carotenoid labs will be one day later than the due date for the paper copies of the lab reports (both are required, of course).

2/27/06:
I've created a turnitin.com section for Wednesday; the password is the same as last semester, but the class number is 1480618 for the Wednesday section. I'll send out an email (which Dr. Chase will authorize going through tomorrow) with the passwords (the same as last semester).

3/1/06:
The due date for the lipid and carotenoid labs has been pushed back to Friday (including on turnitin.com). (Yes, I will be here this weekend, although my time may be limited - as it is this week.) If you turn it in before then, some extra credit will be given.

3/5/06:
Reminder: At least one person from each lab group needs to come in one day in advance (Monday for the Tuesday people, Tuesday after the Tuesday lab has done using the hybridization oven for the Wednesday people) to do prehybridization.

3/7/06:
Several things regarding the plasmid lab and my schedule:

• The plasmid lab is (currently) due March 28th/29th (for the Tuesday/Wednesday labs, respectively). I am inquiring of Dr. Chase as to postponing this for a week or so. The plasmid lab is now due Friday, April 7th for both sections. I will be here that weekend and thus will both be able to help you finish up the lab and accept the labs once they're done, so effectively it's due by Sunday, April 9th, or sometime early in the morning of Monday, April 10th. The date was moved back due to both:
  1. I have a 5-page paper to write up due March 27th for my graduate seminar; and
  2. There is a general biochem exam the week of the 28th/29th.
• I will be available this coming weekend (March 10th-12th) to start work on the plasmid lab (check in 202, including calling extension 202, if you can't find me in my office - I may be using the whiteboards there). After that, until after March 23rd (when I have a meeting with my dissertation committee), I will not be available (outside of lab periods) except for brief questions via email.
• I have a 5-page paper due on March 27th for my graduate seminar. I will therefore not be available the weekend of March 24th-26th, except for brief, emailed questions.
• I then have a presentation for my graduate seminar on April 3rd, so I will not be available on Sunday, April 2nd.

• For the procedural writeup on the plasmid lab, I just want:
  • All numbers that differ from something in the lab manual, especially measurements and amounts of solution - I want to see, for instance, a copy of the table that's up on the board (modified for your amounts if different), your sample's absorption, your sample's fluorescence, what dilutions you used, etcetera.
  • Anything else different from the lab manual, even if we told you to do something differently orally - I may not remember exactly what we said.
  • Anything that went wrong.
  • Anything that went differently from how you would expect, or from how the lab manual lead to you expecting, or that Dr. Chase, Emilia, or myself said was odd, or whatever.
  I do not want a copy of the procedure from the lab manual! I don't want to have to try to read over and grade that much text - for one thing, it'll make it harder to find what I am grading (namely the above).
• I will put up the pictures of the gels (at least for the Tuesday lab, and also for the Wednesday lab if someone tells me which pictures to grab) plus a picture of the standard shortly - ditto for pictures of the blots, once they are finished. Please remind me on this if I haven't done it by Friday or so.
• You can use TotalLab on the gel pictures (and the blot pictures, if it'll work right on those - I have my doubts). We will inquire of Peter Anderson about getting TotalLab demo copies running on some of the other computers if need be. If your lab group wants to try using TotalLab, please let myself and/or Dr. Chase know ASAP so we can tell whether we need more computers to have TotalLab running on them. (The computer in 214 with TotalLab (a non-demo, as in expensive but hopefully fully functional, copy) already on it has been labeled by Peter Anderson (thanks!) on a yellow and black sign.)

As well as the plasmid lab, I will also be grading the sequencing lab, and will further inform you later as to what I expect for it. I am not sure who will be grading the PCR lab and RNA lab (one or both of these may be split up, with one person grading, say, the questions and another person grading the rest of the lab). Laura is grading the lipid lab, and Dr. Chase (as one might guess) is grading the carotenoid lab.

3/10/06:
The Standards for the plasmid lab:

• 1kbLadder.jpg
• 1kbLadder.gif
• 1kbLadder.png

Here are copies of the gel and blot pictures that I was able to find on the computer in 202; I have not had time to do much image manipulation on them as yet (just expand out the contrast range), nor have I had time to convert them into other file formats - I'll work on this. 3/11/06: I'm starting on doing some image editing on the images - refresh this page frequently to see my progress - and am also grouping them by lab group when I can figure that out. Regarding the .combined. files: unless I've had time to do some further image manipulation on them to fix this, due to a bug in the program I'm using (that I haven't had time to find and fix) they do have the problem that the rightmost bit of the picture is moved over to the left. They are in order of the filenames. (Said files are also on the computer in 214 that has TotalLab on it, in the Shared Documents directory. Some of the filenames are a bit different - due to, for instance, that William had saved some of them under names that don't work well on UNIX (with spaces in them) - but you should be able to find it. The ones on that PC have not had any image manipulation on them, so you may wish to download these even to that PC.

• ADS:
  • ads.combined.gel.tif
  • ads.combined.trimmed.gel.tif
  • ads.combined.trimmed.gel.jpg
  • ads.combined.trimmed.gel.gif
  • ads.med.gel.tif
  • ads.high.gel.tif
  • ads.low.gel.tif
  • ads_blot.tif
• det.blot.tif
• DKJKRS:
  • dkjkrs.blot.tif
  • dkjkrs.tif
  • dkjkrs2.tif
• EBEPTP1.tif
• FJC:
  • fjc.combined.gel2.tif
  • fjc.med.gel2.tif
  • fjc.high.gel2.tif
  • fjc.low.gel2.tif
  • fjc.gel.tif
• gillM:
  • gillM.tif
  • gillM2.tif
• gmm.blot.tif
• GL:
  • GL.tif
  • GL2.tif
• hybridized.tm.gl.tif
• Jamie (and lab partners):
  • jamie.tif
  • jamie2.tif
  • jamie3.tif
• MKD:
  • mkd.combined.gel.tif
  • mkd.combined.trimmed.gel.tif
  • mkd.med.gel.tif
  • mkd.high.gel.tif
  • mkd.low.gel.tif
• NGV:
  • ngv.combined.gel.tif
  • ngv.combined.trimmed.gel.tif
  • ngv.med.gel.tif
  • ngv.high.gel.tif
  • ngv.low.gel.tif
  • ngv-blot.tif
• np-AZ:
  • np-AZ-blot.tif
  • np-AZ-blot2.tif
• Neel (and lab partner(s)?):
  • Neel.tif
  • Neel2.tif
• RSJ:
  • Blot:
    • rsj.combined.blot.tif
    • rsj.combined.trimmed.blot.tif
    • rsj.med.blot.tif
    • rsj.high.blot.tif
    • rsj.low.blot.tif
  • Gel:
    • rsj.combined.gel.tif
    • rsj.combined.trimmed.gel.tif
    • rsj.med.gel.tif
    • rsj.high.gel.tif
    • rsj.low.gel.tif
• ST (and lab partner(s)):
  • st.tif
  • st2.tif
• SYS:
  • Gel:
    • sys.combined.gel.tif
    • sys.combined.trimmed.gel.tif
    • sys.med.gel.tif
    • sys.high.gel.tif
    • sys.low.gel.tif
    • sys.combined.gel.gif
    • sys.combined.trimmed.gel.gif
    • sys.med.gel.gif
    • sys.high.gel.gif
    • sys.low.gel.gif
    • sys.combined.gel.jpg
    • sys.combined.trimmed.gel.jpg
    • sys.med.gel.jpg
    • sys.high.gel.jpg
    • sys.low.gel.jpg
  • Blot - combining images didn't work on this one, sorry! I have adjusted the individual images to make it easier to see things on them, however.
    • sys.med.blot.tif
    • sys.high.blot.tif
    • sys.low.blot.tif

Note that, if you can figure out where on your blot your dye front was (e.g., if you marked it with pencil/whatever), you may find it better to do Rfs vs the dye front distance (as in ((distance from wells to band)/(distance from wells to dye front)) - yes, this may turn out above 1) to make your gel and blot more comparable. (On the other hand, if you can see the end of the gel in your blot, then you probably will want to do Rfs vs distance to that for the blot, and Rfs vs distance to the end of the gel for the gel.) Note in this regard that, if you choose to use TotalLab, you should not put the end of the lanes at the dye front, but at the end of the gel or blot, as usual - you then will multiply the Rfs that TotalLab calculates by ((distance from well to end of gel/lane)/(distance from well to dye front)).

3/11/06:
Note: I've done some modifications to the pictures above, including image enhancement, trimming, and creation of combined pictures from the multiple exposure images. I am not finished doing all of them yet, so keep reloading this page if yours doesn't show up as having been done yet. There is currently only one copy of TotalLab running upstairs, since only two lab groups let me know they were planning on using it. However, it is possible to download a demo copy if one is willing to give them an email address - it may have some limits in functionality, however!

3/12/06:
If using TotalLab on the gels from electrophoresis (as opposed to the blots), inverse them! (Dark to light and vice-versa, in other words.) The program is looking for dark spots as bands... (A thank-you goes to Sophia for realizing this before I did!)

3/31/06:
I was sick last weekend (plus another person in my seminar wasn't able to complete typing up his paper), so the due date for the 5-page paper I have to do is now Monday, April 3rd, with the presentation due Monday, April 10th (I don't anticipate that presentation being too much of a problem, however, unless I decide that I need to expand the paper with more references, which is possible). I am available today/tonight and, only for questions answerable quickly, tomorrow (4/1/06 - no April Fool's jokes, please) for going over the plasmid lab, but will definitely not be available anytime Sunday (4/2/06) or Monday (4/3/06) in the morning. Aside from these times (and during the lab itself, of course!), email me for availability. (Sorry that I didn't update this webpage with this info sooner and/or tell y'all about it in class, but I had hoped I could get more done on the paper this last week - I haven't, due to exam grading, other things to get done, plus sometimes still feeling somewhat under the weather.)

4/5/06:
I am grading the plasmid and sequencing focusing labs. I greatly prefer everything but calculations being typed, but do not require it (although it will help subjective credit for people in my section!). I don't want a full writeup of procedure; just tell me all amounts, measurements, deviations from the procedure in the lab manual, problems noticed, etcetera.

For what you need to turn in on turnitin.com for the plasmid lab:

• Description of how you did the restriction mapping - I need to make sure that it wasn't just one person in a lab group doing the restriction mapping (or one person plus me), and the other people in the lab group copying whatever that one person said
• Answers to the questions - not the text of the questions themselves!

I am unfortunately seeing a number of gels in which the 1636 band, despite what it says above in the info about the standards, didn't show up that much more brightly than the rest. One thus has to spot it by how the arrangement of the bands in relation to the well is - above it (closer to the well), there should be one band rather close, a large gap, then a bunch of other bands getting closer and closer together.

BTW, as it turns out I should be available this Sunday (4/10/06). The plasmid lab is due Friday (4/8/06), which in practice means it's due on Sunday.

4/7/06:
Several things:

• You are not required to do the restriction map by putting together circles for each lane (or, rather, for lanes 3-5 and 7-10), but if you find an alternative means, you will need to explain it at least as well as you can putting together lane circles. I find putting together lane circles to work, but I will welcome an alternative means if it also works and you can, and do, explain it. (I also welcome pointers to any websites that give explanations of how to do restriction mapping - it is something that is very hard to communicate!)
• It is preferable to round off the sizes of the gel/blot bands (in bp, not kbp/kb) to the nearest whole number. (Could you have a tenth of a nucleotide? Plus, given the uncertainties of measurement combined with how a log scale tends to blow said uncertainties up, trying to be that precise is silly...)
• I find that people are helped in doing the restriction map if, in their table of sizes for the gel/blot bands (or tables, one for gel and one for blot - either can work, although combining them is, IMO, frequently clearer), they put in for each lane what restriction enzymes were used for that lane (if applicable - for lane 1, this isn't, and lane 6 can simply be called "standard" or "1 kb ladder" or something like that - as long as it's clear, it's fine). This isn't so much for me but for your sake in constructing the restriction map - by this point, I have what enzymes go into which lane pretty much memorized!
• If you have data on the sizes of bands from both the gel and the blot (as in usable data - if your blot didn't work, and you are unsuccessful in getting reasonable sizes from another group's blot, and you state the problem in your lab report, you don't need to do this) you should put in the range of sizes given by each (as in, if you got 1000 for the blot and 1200 for the gel, 1000-1200; or if you got 1250 for the blot and 1025 for the gel, 1025-1250), or otherwise communicate the degree of uncertainty involved.
• The best way I know of to work out sizes for the bands on the blot is to plot blot Rfs (x axis) vs gel Rfs (y axis) for bands that you are pretty sure (given how the pattern of the bands look, how the bands are shaped for large bands, etcetera) correspond, getting the equation of a line, then plugging in all of your blot bands (except for lane 6, which won't give anything useful), not just the earlier-selected ones, to give equivalent gel Rfs. Said equivalent gel Rfs - which represent where the blot band should appear on the gel - can then be treated the same way as your actual gel Rfs to get sizes; in other words, if your standard curve is log(bp) = m*Rf + b (y=mx+b), plug them into the equation of your standard curve (as x), get out y (log(bp)), then take the antilog to get bp. (It is possible that your Rfs for gel and blot will be close enough to not need to get an equation. If so, you're lucky, and can simply use the blot Rfs as if they were gel Rfs. But I have seen rather few cases of this.)
• In regard to the probe, what you are expected to do is to figure out - to the degree possible with your data - where the probe seems to have bound to the plasmid. (You aren't expected to figure out the sequence of the probe!) I suggest doing this by marking each of your lane circles with a circled + where the corresponding restriction fragment (not the entire plasmid) shows up much more strongly on the blot than on the gel, and with a circled - for vice-versa. (Don't put either if the fragment shows up about equally on both the blot and the gel.) You can then transfer these to the final restriction map (for all of the lanes put together) and look at where there are a lot of +'s and not a lot of -'s; this should be where the probe bound.
• The sequencing lab is due at the earliest - it may be postponed - two weeks from this past Tuesday/Wednesday (as in April 18th or 19th, depending on your section). April 28th.
• I will be available this weekend: tonight, Saturday (afternoon/evening), and Sunday (afternoon/evening). The lab is late if it's turned in on Monday after I leave (in the morning) to go to bed or Dr. Chase gets here, whichever happens first. I will be available as late on Sunday night (Monday morning) as I can be, but I do have a seminar in which I'm supposed to be giving an (informal) presentation at 11:35 on Monday, so that will limit me to some degree.
• In regard to the question in the lab about, in lane 1, which size is "true" of relaxed, linear, or supercoiled, just answer which one theoretically would give you the right size (using the standards that we used), even if, for instance, you didn't have a linear band in lane 1.
• You may not be able to see all the bands that "should" be there for a given lane, even using both the blot and the gel. On your restriction map for that lane (if applicable - if you don't have any usable bands for a lane, you can skip that lane for getting restriction maps, so long as you have noted this problem, for instance), you should put in what you estimate to be the size of the section of the plasmid in question - but indicate clearly that it is an estimate. I put the ranges in question in parentheses; you can do however you like, as long as it is clear - parentheses, italicize, use a clearly different font or color, or whatever, as long as it's clear.

4/8/06:
I will be available later on Sunday night than I had originally thought - the seminar I have a presentation for has been rescheduled, for an unfortunate reason (serious illness of a family member of one of the professors).

4/12/06:
Several things:

• For anyone who has not yet completed the plasmid lab, I will try to be available this week to help you with it - email beforehand (or ask me in person, and email a reminder) to make sure I'm available at any given time, however. I should be available this (Wed) afternoon/evening.
• The PCR lab will almost certainly be due before the sequencing lab (See below for when it is now due), since I am grading the latter and probably won't have been able to finish grading the plasmid labs by the time the PCR lab is due (April 18th or 19th); I am also currently still busy with helping people with the plasmid labs and haven't had time to think about exactly what I will be requiring for the sequencing labs. (I'll do my best to get the sequencing labs back at (or, less likely, before) the final exam for this course, which is May 5 at 12 noon, BTW.) So I suggest working on the PCR lab before you work on the sequencing labs. I will endeavour to get up pictures of the PCR gels on this webpage today/tonight - please remind me if they aren't up before tomorrow. If yours didn't work, use someone else's (preferably the other group using the same gel, if applicable), noting which group's results you used (and preferably ask them for permission and for their concentration data - let Dr. Chase or a TA know if they refuse for some reason; if you are unable to get in touch with them, note this, and you can use 0.02 mg/ml for the concentration, with the dilutions as shown on the board in 206). For how much of a procedural writeup will be required for the PCR lab, please ask Dr. Chase if it isn't clear from the lab manual.
• Note that questions should always be worked on seperately, not just written up seperately, except that you can discuss them with Dr. Chase, myself, or another TA in a group (everyone should get the same help, after all).
• For the restriction map, if someone does more than is required in regard to showing that everything adds up correctly (within error), I will give some level of extra credit (not sure how much). If you've already turned your lab in and didn't know about this (I've been telling people verbally but had forgotten to put it up on this page) and thus didn't do this, I apologize; you can have it back for a day or so to work this out if you wish (provided you've already turned in the answers to the questions to turnitin.com).

4/13/06:
Here are the PCR gels, with multiple exposures (and these combined, if combining them worked) when available:

• fjc.ads:
  • fjc.ads.combined3.pcr.tif
  • fjc.ads.combined2.pcr.tif
  • fjc.ads.med.pcr.tif
  • fjc.ads.low.pcr.tif
  • fjc.ads.high.pcr.tif
• grant.jamie:
  • grant.jamie.combined.trimmed.pcr.tif
  • grant.jamie.combined.pcr.tif
  • grant.jamie.med.pcr.tif
  • grant.jamie.low.pcr.tif
  • grant.jamie.high.pcr.tif
• sys.mkd (combining didn't work due to heavy ethidium speckling visible in high-exposure image of gel):
  • sys.mkd.med.pcr.tif
  • sys.mkd.low.pcr.tif
  • sys.mkd.high.pcr.tif
• gill.pcr.tif
• sds.rsj.pcr.tif
• sophia.dan.pcr.tif
• jkldkrs:
  • jkldkrs.pcr.tif
  • jkldkrs2.pcr.tif

4/14/06:
The sequencing lab is now due Monday, April 24th. (The PCR lab is due the usual 2 weeks after its completion - namely, next week, on Tuesday April 18th or Wednesday April 19th, depending on your section. Note that I have put up copies of the PCR gels and standards.)

4/15/06:
The below is in replacement of section 5 in the listing of THE REPORT (page 8) for the sequencing lab (which is due Monday, April 24th):

• Dr. Zylstra put together the sequences. Where did your sequence go in this assembly (e.g., was it the third from the end)? Show the assembly diagram. (This can also help you with section 4 of the report - note the numbers in parentheses beside the sequence identifiers (e.g., 1>315 would say that your sequence was used from 1 to 315).)
• Do a BLASTN using your sequence (not the entire class's sequence; use all of your sequence, even the unclear parts) with the box for "Filter for Lookup Table Only" selected - you may or may not have had this selected earlier - plus the box beside "Low Complexity" should already be selected (if not, select it) - and:
  • List the sequences for which the E-value was 0.0 (there should be some - if not, ask). The sequence identifiers are, essentially, the "gi|#|..." stuff; the sequence descriptions are the text after that. Report both.
  • Report which sequence is from Dr. Zylstra. NCBI is currently not working correctly on BLASTN - see below.
  • Show the alignment (down below the listing of sequence ids) for said sequence from Dr. Zylstra.
  • Give the citation for the paper from Dr. Zylstra (we may come up with some extra credit questions from said paper - I will need to talk with Dr. Chase on this). You can find this out by clicking on the link for Dr. Zylstra's sequence, then looking for the line with "PUBMED #", with the # being a link to the PubMed/Medline record for the (freely available!) paper.
  • For Dr. Zylstra's sequence, there should be more than one protein contained within it. Look at your sequence's alignment vs Dr. Zylstra's sequence. After the material about the E-value, percent identity, gaps (having any vs Dr. Zylstra's sequence indicates a problem with your sequence in that area), etcetera, it has a bunch of lines with Query and Subject (or something like that), with numbers after them, then numbers at the ends of each line. The first number after Subject, on the first Subject line of the alignment, is where on Dr. Zylstra's sequence your sequence started. The last number on the last Subject line of the alignment is where on Dr. Zylstra's sequence your sequence stopped. (The number after Query, on the first Query line of the alignment, and the number on the end of the last Query line of the alignment, is also information for section 4 of the report, BTW, if the region of your sequence that fit was at all unclear; the first would be where on your sequence it started matching the database sequence, while the second number would be where on your sequence it stopped matching the database sequence.) Report this range. Look at the record for Dr. Zylstra's sequence, at the sections with "CDS" (which are links) and ranges of numbers beside them. The ranges of numbers indicate where a protein starts and stops within Dr. Zylstra's sequence. Did your sequence overlap one or more of these? If so, report this, and what proteins these are. If not, report what protein(s) your sequence is before/after.
• Do a BLASTX search using your sequence, with the following modifications:
  • Turn the checkboxes beside something saying "filter" off (no checkmark)
  • Set the genetic code to 11 - for bacteria (mainly of importance for stop/start codons)
  • Set the "expect" to 1
  • Set, in the "Format" section, the number of descriptions and alignments to show to 250 each.
  Once you have done this, for the sequences with, in their alignments, "Identities = #/# (#%)" with the last (percentage identity) number being 65%+ (these are ones that are very similar to your sequence, after your sequence was translated from DNA to protein):
  • List these sequences, including the information from the alignments on the Identities, E-value, etcetera; you need not include the "Query" and "Subject" alignment lines for the BLASTX results. The sequence identifiers are, essentially, the "gi|#|..." stuff; the sequence descriptions are the text after that. Report both, plus the information from the alignments.
  • Give some general idea of what the protein does. For instance, some people have a protein that appears to be an aromatic dehydrogenase. You may be able to tell this just from the lines by the sequence ids, giving a brief description (material in "[]" is usually the name of the species, BTW). Or you may want to click on the links - ones with the format "gi|#|sp|[other stuff]" are especially likely to be helpful.
  Note that some of these sequences may be down below some sequences that have lower than a 65% amino acid identity, due to being short. If your sequence matched to more than one protein when you did the BLASTN search, then it ought to match to more than one protein now. If your BLASTN search did not show your sequence as being within any protein-coding regions (CDS), your BLASTX search should not come up with any lengthy sequences with 65%+ identity (if it does, recheck the BLASTN results, and ask for help if needed) - state this result, and use another group's sequence to do the BLASTX search again - you only need to summarize information on one protein, however, since you're having to do two BLASTX searches.

With regard to section 4 of the report and its question about whether you can see any vector sequence, you only have to answer regarding your own sequence. Answering regarding the entire classes' sequence is extra credit - not much extra credit, since all you really need to do is paste the entire sequence into the "VecScreen" program available via the BLAST webpage and see if anything pops up as significantly resembling your sequence. You should be able to tell from where your sequence is in the entire classes' sequence (aka "assembly", if you want a shorter way to call it) whether your sequence is likely to have vector in it or not. That was a mis-think on my part! Sorry. The best way - aside from VecScreen - to tell whether you may have any vector sequence is by taking a look at the results from the BLASTN search for your sequence. If the ends of your sequence are inside an existing sequence, namely Dr. Zylstra's, which doesn't have any vector sequence in it, then it can't have any vector sequence in it.

With regard to sharing TotalLab data: It is fine for a group to work together on TotalLab and share the resulting data (or for people in two groups to work together and share the data, if one of their group's gels didn't work or whatever, for that matter) - but everyone has to know how to use TotalLab (or, if you've avoided this up to this point, know how to do manual gel measurements). One person doing TotalLab and the rest of the group having no idea how to use it and just using data from that person is not acceptable.

With regard to some of the questions in the sequencing lab:

1. Question 1: Hint: What I say about ddNTP sequencing is "you see where it stops". Does it stop in the primer?
2. Question 2:
   • For both parts of the question, write the position of the phosphate in terms of relative to the base (and sugar) - closest to the base, furthest from the base, or the middle one. Different people think of different ones as being the "first" phosphate.
   • For the second part of the question, what it is asking is which phosphate from the ATP is the one that will be transferred to the end of the DNA. (Hint: If you were the enzyme doing this, which phosphate would be easiest to remove?) That's the one that would need to be radioactive to label the DNA in the Maxim-Gilbert method.

4/17/06:
Note on the PCR lab, section THE REPORT, number 2: This asks for the amount of template DNA in the lane, which is not the same thing as the amount of template DNA that was in the PCR tube (you used 25 ul out of 50 ul in the PCR tube). The simplest way to calculate this is as a part of calculating for number 4 (see below), BTW.

For calculations for number 4, I have talked with Dr. Chase and the following is an acceptable alternative way to calculate the 4 numbers of molecules (one for each of your lanes) that you need to calculate (once for each lane):

• Take the concentration in ug/ul of your stock solution (should be around 0.02 ug/ul).
• Divide it by the dilution factor used for that lane (e.g., 1/10 for the lane from tube #1; if you used the dilution method that is up on the board in 206/207, you can look there for the dilution factors versus stock for the rest of the lanes also).
• Multiply by 5 ul (the amount you put in the PCR tube) to get ug. (You can also take this number and use it for figuring out number 2, BTW - keeping in mind that you need to divide this by 2 before you convert it into ng; don't use this for the next step!)
• Divide by 10^6 to convert ug into g.
• Divide by the molecular weight of the bacterial genome (2.4*10^9) to get moles.
• Multiply by Avogadro's number to get number of molecules of template in the PCR tube.

In regard to the third part of question 2 on the PCR lab: "Why does more time not yield more product, i.e. the final value is fairly constant?" What this is asking is about the temperature the first part of the question was asking about - one of the three of 55, 72, and 94 - and why if you increase the amount of time for it beyond a certain point, you don't get any further increase in product. In other words, this is a temperature for which:

• If you don't give it a minimal amount of time, you get nothing.
• If you give it a moderate amount of time, you get something.
• If you give it a maximal amount of time, you get lots.
• If you increase the amount of time beyond that "maximal amount", you don't get more than "lots" - you just get "lots". But that's still more than you would get for a "moderate amount of time".

In regard to question 1 on the PCR lab, realize that, to get some tubes for which there is apparently no virus present, the solution must be very dilute (relative to the size of the samples). This is essentially a variety of "serial dilution" experiment like in microbiology.

4/18/06:
For the sequencing lab, if you are unsure which file is which, the number from the tube was used as the first number after TC-. If you used another number's sample by mistake (because you looked at the second number, before the .ab1), and have already done the searches et al, don't worry about it. But everyone needs to be sure to make clear which sequence they did searches using.

I have put an assignment up on turnitin.com for the PCR lab questions. The due dates on it are a day after the due dates for the paper copy, due to the lateness of putting up the assignment - sorry about that!

4/19/06:
I will not be available tomorrow (Thursday) until sometime after 8 PM; I will not be available on Friday until sometime after 3 PM - I have a meeting over at DIMACS (Busch campus - yuck!) to go to, and a talk by Dr. Eveleigh for Thursday evening. I will be available the rest of the weekend for help with sequencing labs (and with RNA labs once we figure out what you'll be needing to do for them besides analyzing the gel).

In regard to the RNA lab, Tuesday's worked (in general); Wednesday's didn't. We're still trying to figure out why. I've printed up copies for each group of the necessary data, including data from Tuesday for the Wednesday class - Dr. Chase has these, if they haven't been given to your lab group already. I'll post up (on here) the RNA gel pictures from Tuesday sometime this afternoon/evening - please remind me if I forget.

Here is the Wednesday picture, from a week ago, in a couple of different versions:

• Wed.good.RNA.trimmed.inverted.tif
• Wed.good.RNA.trimmed.tif
• Wed.good.RNA.tif - this one is mainly for seeing that some stuff seems to have travelled off the end of the gel

The Tuesday one, with multiple combinations - I'm not sure what worked the best:

• Tues.good.RNA.combined.low.trimmed.tif
• Tues.good.RNA.combined.trimmed.tif
• Tues.good.RNA.combined2.trimmed.tif
• Tues.good.RNA.med.tif
• Tues.good.RNA.low.tif
• Tues.good.RNA.high.tif
• Tues.good.RNA.high2.tif

4/20/06:
The RNA standards: RNA.standards.Promega.G319.gif

In regard to the quantification of the cDNA, as well as figuring up the cDNA from the 6 numbers you have (Actin and other) for the number of cycles for the non-control (with template) lanes, you will need to figure up the cDNA (in pg or whatever only, not anything more) from the control (no template) values, then subtract that from the amount of cDNA you figure out from the non-control values for the same substance (Actin or other). This will compensate for any cDNA showing up (due to contamination) from your no-template controls.

You do not need to do very much of a writeup for the purification procedure for the RNA lab. Most important will be the figuring up of your concentrations and, if it didn't work very well (at the stage of extracting RNA), trying to figure out why. (Don't worry about figuring out why the Wednesday lab qPCR didn't work - but if you have any good ideas, please do tell us!)

4/20/06:
NCBI is messing up on BLASTN (and maybe BLASTX, I haven't checked yet) searches - it isn't showing Dr. Zylstra's sequence for some reason. If you do a BLASTN search and Dr. Zylstra's sequence doesn't show up (among the ones with an E-value of 0.0, preferably), just use the first one (with multiple proteins in it - multiple "CDS" lines) as if it were Dr. Zylstra's sequence. Let me know if the same thing happens with BLASTX - it shouldn't.

On figuring up the moles mRNA per mg/ug of RNA isolated:

• Do it as moles (or femtomoles or attomoles, if you prefer, as long as it is clearly marked) per ug of RNA, not mg (mg, on page 10, is a typo)
• The instructions on page 10 regarding "this fraction" are unfortunately unclear - Dr. Chase meant "this fraction" in the sense of "this portion" or "this aliquot", not "this proportion". In other words, divide the moles of mRNA by (([amount, in ul, of liquid from the cDNA synthesis reaction used]/[amount in the cDNA synthesis reaction in ul - this should have been 20 ul])*[total amount, in ug, of RNA put into the cDNA synthesis reaction - this should have been 1 ug]).

I have (in my office) the qPCR printouts for groups F (row F, from Wednesday), group E (row E, from Wednesday), and group 4 (column 4, from Tuesday). I also have a couple of spare standard curve graphs for sucrose synthase.

4/22/06:
For the RNA lab, the size of 25S rRNA for Arabidopsis is rather hard to find; it's 3375 bp. For the size of 18S and that of tRNA, see your general biochem textbook.

Remember to do turnitin.com for the PCR lab! And I will create a turnitin.com thing for the sequencing lab questions; ditto, probably, for the question on the RNA lab - I will have to check with Dr. Chase on this.

For the Tuesday lab bp standards (on the RNA gel), and possibly for the Wednesday lab standards: Note on the RNA lab that you will almost certainly either need to fit the standards using SigmaPlot and the equation that Dr. Chase gives in the lab manual, or using two lines (one for one part of the graph and the other for the other part of the graph), or do it manually with semilog graph paper. Correction: The equation given in the lab manual will only work for the top part of the graph, and is probably not necessary if you delete the top one or two points. You should be able to fit the graph best either with two lines (one for the first few points on the graph (for which you could use the equation in the lab manual), one for the last few points on the graph, with at least one standard point included on both) or with a manual fit using semilog graph paper (only advised if you are good at this! I wouldn't do it myself...).

For the RNA lab calculation of the ratio between the other gene's results and the results from actin, you will need to do this in terms of moles (more likely, in terms of femtomoles or attomoles), not picograms/nanograms/whatever, since the different mRNAs differ somewhat in molecular weights. (Technically, instead of multiplying the number of base pairs by the average molecular weight of a base (which should probably be by the weight of a base pair - I am checking with Dr. Chase on that), we should be using the molecular weight of the mRNAs (which we can calculate from what the sequences are), but that would require my digging up the sequences again...)

For interpreting the BLASTX results, note that catechol is aromatic.

With regard to .tif (TIFF) format pictures on this website, such as the RNA lab gel pictures, you may find that it will work better if you save them to a memory stick (flashdrive) and open them from there (a thank-you goes to Sophia for this tip). Another technique that can fix some problems is, if you can open them in Photoshop Elements (or Photoshop), save the image as a TIFF (.tif ending, again) and, when it asks something about "IBM/PC" vs "Mac" formats, save it as the former.

4/23/06:
If Dr. Chase checked the absorbances for your RNA sample, then the dilution factor was 200, not 100, as it turns out. I am not sure if the dilution was modified for samples that were run on the spectrophotometers in 206/207.

I have put up an assignment for the sequencing lab questions on turnitin.com. I suspect I will likewise be doing so for the RNA lab question, but will have to check with Dr. Chase before doing so.

I am likely to be occupied next week after Monday with grading the plasmid labs, at least until Friday, but am not sure as to my exact schedule on this; I will try to post up more definite information as to my availability later. Dr. Chase (and perhaps Laura) will be grading the RNA labs, so after Monday I suggest asking Dr. Chase questions about the lab if he's available (and emailing him your questions if neither he nor I are available and you can't find Laura). I think I will try to be available Tuesday after 2:00PM or so - not sure about the rest of the week. Sorry to the Wednesday lab people, but there's no way I would be awake enough Wednesday morning to help y'all out...

4/24/06:
If you haven't gotten to that stage yet on the RNA/qPCR lab, you do not need to subtract the picograms (or nanograms) from the no-template controls from the experimental values. It's fine if you've already done so, however. (We aren't sure whether it's a good idea or not...)

In figuring out the amount of total RNA in the stock solutions (in the first week of the lab), you can assume that they had a volume of 20 ul.

In figuring out the number of moles (or picomoles, or femtomoles, or attomoles - any of these is fine, as long as you label it properly) of cDNA, you should divide by 2 to account for that 305 is a molecular weight for bases, not base pairs. However, if you have already done this calculation following the lab manual, don't worry about it.

The due date on the sequencing lab is still today, 4/24/06. The reason that turnitin.com says 4/25/06 for the questions is that it's set to 4:00AM, so people turning things in while I'm still likely to be here accepting them as in on 4/24/06 won't be indicated as late on turnitin.com (I'll take a look at the dates and times manually, of course, and won't count things as late if I'm still here after 4:00AM (I was, BTW)).

4/25/06:
For the qPCR lab, if your "anaerobic gene" was ADH (alcohol dehydrogenase - for which one, if you're curious, ask Dr. Chase):

• There are two lines on the graph. Use the BioRad one. It corresponds to the equation that has a continuous line beside it (the one with the lower number at the start - that would be the Y-intercept if the graph weren't log-scale).
• Yes, it's cycles vs nanograms, not picograms. Apparently there's an awful lot of ADH mRNA...

With regard to question 1 in the sequencing lab, it is asking whether you will see the primer's sequence in the sequence you get from a Sanger dideoxy nucleotide sequencing reaction - not whether, for instance, you would see it in a sequence assembled from multiple sequences (e.g., the class' sequence ("contig") in our case).

I will only be available on Wednesday and Thursday via email, and my response time is likely to be rather slow even for email. Do not disturb me in my office while I am grading (unless you work in Dr. Kahn's lab and are needing something related to research or to use the computer in 119). On Wednesday and Thursday, I advise checking first with Dr. Chase regarding the RNA/qPCR lab if at all possible.

With regard to the RNA/qPCR gel, you only need to analyze your lane(s) and the standards, not the other lanes.

4/28/06:
The final exam is at May 5 at 12 noon, in room 106 of the Douglass Chemistry building (aka Regina B. Heldrich Science Building).

I am semi-available today (I'm trying to do grading on the plasmid lab - be forewarned that grading tends to put me in a bad mood...), but recommend email as the way to get in touch with me for questions if at all possible. I am, of course, available to hand labs into.

A reminder on lab questions:

• WORK INDEPENDENTLY - except that it's fine if multiple people hear what Dr. Chase or a TA says, since everyone should get the same help
• CITE YOUR SOURCES - if you take anything from online (other than this webpage), from a book (other than the lecture textbook), etcetera, you must cite your source; this is not just our policy, but Rutgers (and academic in general) policy (vs plagarism) About the only exception to this is simple factual information (not in the form of a direct quote) that is available from thousands of sources - sizes of rRNA or tRNA, for instance, or the "standard" genetic code.

I would appreciate it if someone would remind me of who got the computers to produce those nice circles for plasmid mapping, BTW.

There will not be anything on turnitin.com for the RNA lab. People who have not yet turned things in on it should do so ASAP; given my lateness in putting up assignments on there, we haven't really been counting off for lateness on turnitin.com this semester, but there is only so far that that will go!

With regard to the plasmid labs, I'm working on grading them (the PCR labs are somewhat graded - by William - but need some revisions). Samira (thanks!) has given me a site for people to take a look at that should be useful in reviewing how to construct a restriction map (especially for those who haven't had Molecular Genetics), namely http://www.carolina.com/biotech/plasmid_problems/plasmid_guide2.asp.

4/29/06:
I didn't get much sleep last night (had trouble going to sleep then woke up and had problems getting back to sleep), then was up at 8:15 (later than I'd intended...) for Ag Field Day. I'm going upstairs to 325 (unless it's in use, which I doubt) to lay down for a while. Ah, well. Some alumni meeting or another seemed to be scheduled in there - or at least lots of people coming by thought so. More stimulants for Allen instead of sleep...

5/1/06:
I'm going to try to get some more grading done, so DO NOT DISTURB. If you've got an RNA lab to hand in, try Dr. Chase, or if he's not in, get a secretary or professor or grad student or whoever (not another undergrad) to sign and date it, then stick it in Dr. Chase's box. If you need to ask questions about a lab report and Dr. Chase isn't available, you can try email, but my response time is likely to be rather slow (and for the RNA lab, since I'm not grading it, I recommend emailing Dr. Chase first anyway).

I will be in until sometime between 10:00pm and midnight. Yes, you can give labs to me to sign if nobody else is available - under my door is preferred, however, if it will fit; if it won't, then you can knock or whatever. I will try to remember to check my mailbox before I leave (that's the box across from Eileen's office, not the box for old exams outside my door), but I may forget.

5/2/06:
DO NOT DISTURB. I am beginning to doubt whether I'll have the plasmid labs graded prior to the exam - much less the sequencing labs at the exam - but I'm trying.

5/3/06:
The plasmid labs are graded and are in Dr. Chase's office, since I'll be at the 404 exam (8AM... groan!). People generally did well, although at Dr. Chase's request I limited myself to fewer extra credit points than for the electrophoresis lab last semester (sorry!). The high was 116; the low was 52; the mean was 96.9; the median was 99. I'll try to do the sequencing labs tomorrow afternoon/evening and maybe Friday morning, although I'm also going to try to create a plasmid mapping question (same sort of thing as in past years, but varying per person, just as are the sequences for the sequencing question I've written).

5/4/06:
The plasmid labs are available from me in my office. Do not disturb except for getting these; I'm grading the sequencing labs currently and exam grading is going on in 119. Notes regarding the final:

• The final exam is at May 5 at 12 noon, in room 106 of the Douglass Chemistry building (aka Regina B. Heldrich Science Building).

• I normally give a sequencing question on the final exam, and will probably give a plasmid question (which will in most respects be similar to the ones from past years). I have put an example of the sequencing question I generally ask in expbio.question.example.txt; it also includes the grading master. Note that the sequence in the question (and thus the master) varies from person to person, and that the question itself might vary from the example (probably only slightly, though).
• For past final exams, see http://www.iris.rutgers.edu/iris.html, go to "Reserves", put in "Chase", click on "Chase, Theodore", then scroll down to where it starts saying "Experimental Biochemistry Spring ####" (where "####" is the year) or something like that, then click on "Details", then click on the link to the PDF file. The old exams are unfortunately not currently reachable from Dr. Chase's webpage - I am working on getting this fixed, but am unfortunately not the one who maintains Dr. Chase's website. If you are unable to get the old exams from Dr. Chase's webpage (http://aesop.rutgers.edu/~dbm/tedchase.html), try using a different computer and/or different web-browser (e.g., switch from Internet Exploder to Firefox).
• One thing that people keep getting messed up on on the plasmid labs is thinking that they can say that a restriction enzyme cuts at, say, 2000 bp because they have a 2000 bp fragment. That is only the case if you have some means of determining a 0 point and also can tell that the fragment in question starts at a site at that 0 point (and goes to a site 2000 bp away from that 0 point)! And note that that 0 point is also numbered equal to the size of the entire plasmid. I have seen a total of one person manage to get this right, and multiple people who got completely confused by trying to number the base pair positions. Think of it in terms of lengths between sites, unless you're the one person who managed to get it right using base pair positions. (The manual gives base pair positions for the vector because we have the full sequence of the vector - not from restriction mapping of the vector, so far as I know!)
• Similarly, if you have two enzymes cutting and producing two fragments, then you do not have one fragment from enzyme A and one fragment from enzyme B - both fragments are from both enzymes!

5/5/06:
Several things regarding the final:

• Be forewarned that I may put more restriction enzymes into the plasmid problem for the exam than has been on it in previous years!
• However, I am also not going to make you compute sizes from cm or Rfs. Dr. Chase may well have you doing a graph on the exam on another problem, however!
• See above for more info.

I am afraid that I haven't gotten all the sequencing labs done, due to working on the plasmid question for the final (I spent from ~4 AM till 10 or 11 AM today doing so). However, exactly why the plasmid question took me as long as it did should be comforting to students - namely making sure it isn't overly difficult! It is less complicated than it looks in terms of solving it, if more complicated than it looks in terms of creating it...

Comments

People frequently say that the lab takes more time than it should for the number of credits. You are correct; Dr. Chase and I agree with you. Unfortunately, it appears to be University policy, probably due to the various humanities departments lobbying, to not count laboratory hours as much as classroom hours - even if the laboratory in question has, like Experimental Biochemistry, lab reports that require lots of time outside of class. On the other hand, do realize that this is one of the more thorough biochemistry (and related areas) laboratory courses that undergraduates might ever take, and definitely gives people lots of experience - experience that has meant the difference between getting a job and not getting a job for some. (The course credit hours have been increased a bit via adding extra time to the lecture, plus combining the course with Data Treatment, but it's still too few credits in my opinion.)

I have suggested to Dr. Chase that the Rutgers Genetics course (as variable in quality as I've heard it is - some report it being better-taught in the summertime, BTW), or some equivalent, be prerequisites or corequisites for the second semester of Experimental Biochemistry - and may suggest this also for the second semester of General Biochemistry - in light of the many people coming to me needing help on what I, as a geneticist, would consider very basic aspects of the plasmid, RNA, sequencing, and PCR labs. While this would take too much wrangling to get through for next year, he is planning on adding a strong recommendation for such a course to the description of Experimental Biochemistry in the course catalog.

I am willing to spend quite a bit of time giving assistance, as you can see from the scheduling comments above. Some (as expressed in one anonymously-made comment) may be concerned about whether or not the people I work with are doing enough on their own:

• When I work with people, I want them to do it right. This has been known to result in doing somewhat more work than the lab manual may technically call for (although this also means that, at least if I'm grading it, the person can get more extra credit if it's done right).
• Learning styles are quite variable. Sometimes, it takes telling people things several different ways for them to get it. Sometimes, it takes leading them through how to do something before they understand it.
• If someone is asking questions instead of thinking (instead of asking questions to check their thinking, or because of a lack of clarity in what appears to be required or in how the procedure is supposed to work, or because they've struggled with trying to understand something but, perhaps because of the different learning styles I just mentioned, just can't quite get it), then:
  • This won't help the person on the exam - it'll hurt them.
  • People who don't understand what they're doing and are just following directions tend to:
    • get things wrong and either get points taken off or have to go back and correct the errors, thus doing more work; and/or
    • not do a very good job of discussing their results and answering the questions, thus getting points off there - and, at least if someone's in my lab section and it's a lab report I'm grading, then how well you discuss your results and answer the questions are a factor in your subjective grade.
  • I've gotten pretty good at not showing it, but I do have a temper. Failing to think is an excellent way to get me irritated.
  Please do not let the above make you less likely to seek help from me when you need it. While I am indeed irritated when people fail to think, I find it just as irritating when people fail to ask for help when they need it and I then have to deal with the results, either in grading or in a laboratory procedure.

Intro Sheet

My background: My primary background is in biology, specifically molecular genetics. I am mainly qualified for this lab due to:

1. prior lab experience, especially with DNA, electrophoresis, etcetera; and
2. having TAed it before (this will be my 8th or so time for the fall labs).

Getting in touch: The best means of getting in touch with me is to come by Lipman Hall room 118/119, then try Lipman Hall 202 (the SGI computer lab). (If the building is locked up, try the phone number given below - use the 119 number first in that case.) The second best is to email me (see below for the address), since I check my email several times most days. (Note the points on my tutorials page about not sending me email that's something other than plain text.) The third best is to call me at 932-9255 extension 119 (202 if that doesn't work; 207 if at night and neither 119 nor 202 have worked). (Do not assume that I'll receive voicemail; only use this method if I (or someone else) answer(s) the call.) The fourth best is to put a note in my box; it is on the first floor of Lipman Hall. (By the way, if you are turning in a lab report other than directly to a TA or to Dr. Chase (preferably not into a mailbox - try under the door of Dr. Chase's office), be sure to get someone - a secretary, professor, graduate student, whoever, just someone other than another undergraduate - to sign and date it so we don't have to count off for lateness (or for any more lateness than you should have been counted off for).

Office Hours: I will try to let you know what times I will be available (usually I'll be in and available a lot immediately prior to when each lab report is due); the best thing to do is to simply ask whether I will be available at a given time - people who've had me (as a TA) before can tell you that I am willing (unless other obligations, including my own academics and my need for sleep, conflict) to work with people at quite odd times and/or for very long hours.

Quizzes: My quizzes have as their primary purpose encouraging you to have read over the lab before you come in and making sure that you know, in particular, safety-related information. I do not expect you to have memorized all of it; my own memory isn't that good, and I will generally consult the lab manual before answering questions - but I will have read over the lab. I expect you to know in general terms what we are supposed to be doing that day and about any safety precautions that you need to take, particularly those which can affect other people. I may - I generally don't unless I get inspired or feel that you aren't reading over things - give you a take-home, open-library quiz (or at least a question or two, if not a full quiz) that is for the next week's lab, again mainly to encourage you to read it over. Except for take-home quizzes and safety quizzes, I will not ask for any further quiz-taking.

Subjective Grade: How I do the subjective grade is to note down when you do something good, and when you do something bad. I will fix a particular starting grade, and doing something bad will decrease your subjective grade below this; doing something good will increase it above this. The starting grade and amount up/down will be determined by whatever gives a final mean of 85 and a high of 100. Examples of good and bad things:

• Good things:
  • asking an intelligent question;
  • giving an intelligent answer on a quiz;
  • giving an amusing answer on a quiz;
  • typing a take-home quiz;
  • typing lab reports, particularly the ones that I grade (which are the gel electrophoresis and isoelectric focusing ones for the Fall Semester) - but I do not expect you to type calculations!;
  • doing significantly more work than other people;
  • giving a good answer to the questions on a lab that I grade;
  • helping someone else in the lab (especially someone not your lab partner);
  • cleaning up messes that you didn't create;
  • doing extra work because of things that aren't your fault;
  • pointing out problems to us (especially if you provide the solution also);
  • for the Spring Semester, choosing to do the Carotenoid lab (generally considered harder but more interesting - there have been exceptions to this, depending on sources of material chosen, however; I recommend nuts as a source for Lipids) if most people are choosing to do the Lipid lab - or vice-versa, if that happens
  • for the Spring Semester, using a material I consider interesting (most especially anything I purchased!) for the Carotenoid or Lipid labs
• Bad things:
  • asking a question that makes it apparent that you haven't read over the lab (having read over it and not understanding it due to writing style, lack of clarity, or whatever - different people understand different ways of putting things, after all - is not, however, a problem at all, and if it leads me to make suggestions to Dr. Chase as to how the lab manual can be improved, can get you an increased subjective grade);
  • doing something that makes it apparent you haven't read over the lab (more off for this than the first - if you don't know something, do ask);
  • coming in more than 15 or so minutes late without an adequate excuse (this may also result in your missing a quiz - which you will not be able to make up without a good excuse for coming in that late);
  • leaving a mess that I or Emilia have to clean up (particularly the latter, if she has cause to complain to me);
  • doing significantly less work than your lab partners;
  • doing something that irritates your lab partners (unless you and they come to me and/or Dr. Chase and explain the problem and we decide you're in the right);
  • Putting extreaneous material into turnitin.com so that you have "false positives" on your turnitin.com results (plagarism indications that aren't false will have rather worse effects!)

I normally find I have more +'s than -'s by the end of a semester. This means that those who do get significant minuses (e.g., a lab group a bit back that left lots of gunk in the pig kidney centrifuge bottles...) will get a rather low subjective grade, and that those who simply don't do much either positive or negative won't get a particularly good one.

Nametags: I have problems remembering people's names (including close friends and relatives, BTW!), especially in a class of 20+ people. This sometimes causes problems with assigning subjective grades. Something I'm trying out this year is to have everyone fill out and wear a nametag. These should be left in the lab, to avoid losing them. At least for the fall semester, if you forget to wear it, that will be subjective points off; if you lose yours, that will be even more subjective points off especially since I bought them with my own money!

Curving: I normally will try to curve to a mean of 85 and a high of 100 (although I normally do not curve down). On lab reports and quizzes, I'll circle the final grade that you'll get for each of them. Such curving does not include extra credit points or points taken off for lateness.

Page written by Allen Smith (send mail to meatcan2@beatrice.rutgers.edu, substituting easmith for meatcan2 - do not just use the mailto link! - see my antispam page(s) for why).

I am not responsible for any pages linked from these, except for those that I have written. Neither is the Structural Biology Computational Laboratory, the Department of Biochemistry and Microbiology, Cook College, or Rutgers University responsible for (or have any copyright on) pages that I have written. My webpages are not official Rutgers webpages.