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My Teaching: TAing Experimental Biochemistry

I TA one of the labs in the course Experimental Biochemistry (note that this description needs updating; also see the spring semester course description), with the professor for that course being Dr. Theodore Chase, including supervision of students in the laboratory, helping students with lab reports, grading of some lab reports, and helping grade the final exams. Below, you will find:
  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.
For the 2001-2002 web-page, see For the 2002-2003 web-page, see For the 2003-2004 web-page, see For the 2004-2005 web-page, see I am afraid the below is somewhat out of date, at least with regard to the Intro section and below it.

Information for Current Students

We are not requiring prelabs, at least for the Tuesday labs. Nor do I plan on requiring them for the Tuesday labs next semester.

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.

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.

In regard to, 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! 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 account, talk to Rob Muldowney in Lipman Hall 202 (or email him at or 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 (namely, the questions in the pH lab).

We have wound up sending out the 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")!

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.)

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 - sorry, I had thought that it was already set up!

What you submit from the pH lab to 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.

You should only submit the answers to the questions, not the questions themselves, to You should not try to submit anything that is not text - no figures/drawings or calculations. You can submit the answers to 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 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 version for, first, plagarism, and second, for whether it is the same as the version with your lab report. For getting a account or for help using said account, consult Rob Muldowney if at all possible.

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.

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).

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 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 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'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 usage between General Biochemistry tutorials and Experimental Biochemistry lab reports:

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!):

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).

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!):

Protamine sulfate is notable for being high in arginines and, to a lesser degree, low in tyrosines and tryptophans. Ignore the sulfate part for purposes of reactivity in various methods.

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:

This is particularly likely to be necessary for the point with the smallest amount of protein (remember the pipetting/dilution lab?) and/or for points with an absorbance above 2 (for which the spectrophotometers will be getting inaccurate); for the Coomassie Blue data, see below. But it may not be necessary in your particular case - look at the data and decide. (One way to tell: if the R-squared value for the line is less than 0.95 or so, you probably need to take out some points. If the R-squared is greater than 0.99 or so, you probably do not need to take out some points. But you have to judge based on the situation.)

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.

We now have a policy for what happens if you turn a lab in on a given date, but do not turn it into 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 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, 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 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 E1 mg/mL for the protein determination procedure, here is the easiest way I know of to do it:

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.

Several things:

(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.

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").)

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.

Four things for the statistical workup:

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).

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.

Three things:

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.

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).

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!))

A few things on the carbohydrate lab:

Two things:

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.

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.

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

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...

Several things (some important for all students in the course!):

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:

Dr. Kahn and Dr. Chase both say that one should be able to get rough estimates for Km and Vmax (good enough for using for initial values for a nonlinear curve fitting) even without a linear-form plot, by just looking at one's values or a graph of them without a curve fit, but I know that I have problems doing this, and even they would need a linear plot if the enzyme did not get near its Vmax (due to, say, inhibition or the high-substrate-concentration points having to be removed due to problems with them - for the latter, most likely because the substrate being plotted is D-alanine but the enzyme runs out of another substrate, oxygen (see extra credit experiment 11)).

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!

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

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.

Reminder: You also need to submit the procedural writeup and the answers to the questions, in separate files, to 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 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.

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

The way to figure out that this is happening is to look at the spacing of the numbers along the bottom - 0, 0.005, 0.01, 0.02, 0.04, 0.1, for instance, should not be spaced equal distances from each other! This tends to result in what looks like a curved line, BTW.

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 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...)

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.

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.

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.

For wednesday, putting together the different exposures worked weirdly but, in my non-humble opinion, rather well for the 3-9 pH range one, but I have to admit rather weirdly in general for the 4-6.5 pH range one. The standards are as follows (sorry about lack of a picture, but the idiots at BioRad locked the PDF so that the picture isn't extractable, and I refuse to provide them with free advertisement by giving the link to the PDF; I can probably do a screen capture to get the image if people really need it); the below run from high pH to low pH (and thus should be cathode to anode, unless I'm so groggy I'm mixing them up...): The above run the with the top being next to the tab; see below!
Listing for Tuesday of whose sample is in what lane:
  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)
The lanes run from 1 at the bottom of the gel in the pictures to 8 at the top of the gel; I know this because there are 3 lanes below the standards and 4 lanes above the standards. Listing for Wednesday of who is in what lane:
  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)
Again, the lanes run from 1 at the bottom of the picture to 8 at the top.

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.

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.

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.

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

To clarify on what is required in regard to gel analysis: Amounts of extra credit for various things will vary depending on how many people do said extra credit. (You get extra credit only if something is done correctly and thoroughly.)

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:

Do not simply tell me the amounts that the lab procedure asks for, except for the standards. What matters most is what you did, not what you were theoretically supposed to do. Only reference what the lab manual says for amounts of protein or activity to explain why you used the amounts of sample that you did (or in order to explain why yours didn't work, if you didn't follow the directions!).

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.

Three things on the questions:

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.

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.

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:

See whether the two ratios are approximately similar. If so, state as much. If not, try to figure out why not (for instance, was there a problem with your after-dialysis specific activity? are you identifying the right band as D-amino-acid-oxidase?). It is good but not required for you to comment on how many bands are in the final enzyme lane vs the dialyzed precipitate lane (hopefully, the final enzyme lane has fewer!).

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.

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.

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.

The departmental website is down; see:

for some old exams (most, possibly all with answers) I've been able to locate via Google's cached pages. I've been able to contact Dr. Chase after struggling with idiot Verizon's directory assistance nuttiness (gods-damned monopoly!); he had taken home almost all of the paper copies of the 2001-2004 exams for reference while creating the upcoming exam.

The departmental website appears to be back up; see!

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!)

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!

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.

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

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.

The test gel for the Tuesday section, with formats as per the filenames:

From the bottom up, the lanes were:
  1. NEV
  2. SV
  3. MKD
  4. SYS
  5. RJF
  6. JYX
  7. CJF (B)
From these, the people in lanes 3 and 5 (starting with 1 at the bottom) have both genomic and plasmid DNA (perhaps half and half for lane 5, not sure for lane 3 given the "smear"). The groups in lanes 2 and 5 do not appear to have much RNA, which hopefully is what your fluorescence vs absorbance measurements likewise indicate.

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

Yes, we realize that some aspects will be the same within a lab group - numbers, what you think you have, etcetera. We will be giving you an extra day to turn things in to

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:

We did not run seperate standards - in following the instructions in the lab manual to graph log(retention time) vs number of carbon atoms, use whatever appropriate fatty acids (#:0 for saturated, or #:1 w9c for monounsaturated omega-9) you find on your GC results.

I've created the Tuesday section on; 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 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).

I've created a 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).

The due date for the lipid and carotenoid labs has been pushed back to Friday (including on (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.

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.

Several things regarding the plasmid lab and my schedule:

There are some updates to the above - see below.
Regarding the plasmid lab itself:

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.

The Standards for the plasmid lab:

The brightest band should be the 1636 band; the 506/517 band should be the second-brightest after that, unless it's covered over by the dye marker.

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.

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)).

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!

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!)

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.)

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 for the plasmid lab:

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.

Several things:

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).

Several things:

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

I may also see about making copies of all of them in other file formats if people have problems with the file format of these (let me know if so). William will probably be grading the PCR labs, BTW. The PCR standard should be a Promega 100bp ladder; the brightest band should be the 500bp band.

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.)

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):

For section 5, figuring up anything using the entire classes' sequence other than where your sequence is in it will be extra credit - this would mostly involve analyzing the results of BLASTX searches using the entire sequence, the same way as above.

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:
Also note that I normally give a sequencing question on the final exam which is at May 5 at 12 noon this year, and plan on doing so this year. (A plasmid mapping question may be from me or may be from Dr. Chase, but is also likely in any event.) 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).

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):

It is also OK, of course, to do this calculation the way mentioned in the lab manual (with molarity) - you should get the same result.

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:

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.

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 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!

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:

I will have to check on exactly what the standards are - Emilia has that info in her office - but I thought I'd put these up so that people could start on measuring the bands. I am not sure where the Wednesday picture from today got saved - I'm pretty sure it was saved, however; I will try to check on this. The Tuesday one involves multiple files to put together, so will take a bit longer.

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

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!)

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:

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.

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 for the PCR lab! And I will create a 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.

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 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...

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 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 (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)).

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

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.

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:

We will at the minimum take off for failure to observe these, and have previously and may well again in the future bring people up on judicial charges for severe (including any repeat) offenses.

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 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 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

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...

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.

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.

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).

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:

Several things regarding the final:

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...


I am not enthused about the amount of memorization required for the final exam. (Dr. Chase is not of the opinion that it is that heavy on memorization - but he has an extremely good memory, so perhaps does not fully realize how difficult memorization can be, especially for those of us like me who have a bad memory. Incidentally, I recommend studying old exams as the best way to do OK on the final - see for old exams with answers (at the bottom of the page), plus check on IRIS ( in the online reserves using Dr. Chase's name. For the spring semester, I generally put together a question (either on plasmid assembly or on dideoxynucleotide sequencing).) My disapproval of memorization where not absolutely necessary is one reason that I don't give in-class, closed-book quizzes except on safety matters - things that people do need to know things off the tops of their heads, to avoid endangering anyone. (I have a particular concern with regard to harming other people - speaking from my political/ ethical viewpoint, if someone harms themselves when they knew or could have known if they'd bothered to find out the danger, that's their business (I find it unfortunate - I don't like seeing people harm themselves - but freedom comes with responsibilities). Unfortunately, the legal system in the US, and other governmental regulatory means in most of the rest of the world, don't agree, and I do try to avoid either getting other people in trouble or fighting fights I can't (yet) win.)

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:

Intro Sheet

Tuesday Lab

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:

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.

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