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My Teaching: TAing Experimental Biochemistry - 2002-2003 webpage

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

Information for Current Students

9/30/02:
The Protein Determination lab is due on October 11, 2002; I am grading it, so please type it. It is due this much later than normal because Dr. Kahn will be lecturing that week, in the lab lecture, on how to use SigmaPlot to do line-fitting, so we concluded that people should be given some time after that to benefit from said lecture when doing the lab report. (It is fine if you do the line-fitting via Excel or another program, BTW.)

11/20/02:
Here are (most/all of) the SDS and native (activity-stained) gels from the Tuesday lab. I'll do some image reworking/improvements on them after I've also got the ones from the Wednesday lab (and after I catch up on sleep... note that I will be available Wednesday from 12:00 or so to at least midnight to help people with the enzyme lab; I apologize for not having the protein labs back yet).

• jim-steph-banks.trans.coom.jpg
• jsb.coom1.jpg
• native_gel_rf_jc_cb.jpg
• LLMnativegel.jpg
• sds.yiannos.work.tif
• yiannos.coom.tif
• brianlab5.jpg
• deelab5.jpg

12/8/02:
Due to finding myself falling asleep at the keyboard, I had to go home and sleep for about 12 hours (longer than I had intended - my alarm didn't wake me up...) - I apologize to anyone I missed seeing due to this. I'm now here and available to help with the labs. Gavin has not yet given me the pictures for the isoelectric gel to post up. The due date for the lab will be pushed back by at least a day, more for the isoelectric focusing part (although this means I may not have them back at the exam, I'm afraid). Here is some info on the standards (unstained - without the different colors - until you put the Coomassie Blue on them) used in the SDS gels (THIS IS DIFFERENT FROM THE INFORMATION EARLIER - SORRY ABOUT THAT! I had not gotten the info from Emilia that I thought I had...):

• Pictures (3 different formats - .tif is preferable, but is large and not usable by all browsers):
  • sds.standards.tif
  • sds.standards.jpg
  • sds.standards.gif
• Molecular weights:
  • 250,000 Daltons
  • 150,000 Daltons
  • 100,000 Daltons
  • 75,000 Daltons
  • 50,000 Daltons - Darker band
  • 37,000 Daltons
  • 25,000 Daltons
  • 15,000 Daltons
  • 10,000 Daltons

12/9/02:
Below are the Tuesday and Wednesday Isoelectric Focusing gels. (This would have taken less time if Gavin had sent them to me via some other means than email and in some other format than PowerPoint... imagine trying to read such an email on, say, eden.rutgers.edu. Gavin is still to be thanked for his help on this, however - none of us realized the headache that happened with the image transfer, and it seems to have been caused by how he was improving the pictures with info (like the pIs of the standards!) that you'll need.) The gel picture (gels from both classes - for "Thursday", read "Tuesday"), in 2 different formats:

• ief.gel.jpg
• ief.gel.gif

• All of the lab but the Isoelectric focusing part will be due:
  • Tuesday People: Wednesday, Dec 11th
  • Wednesday People: Thursday, Dec 12th
• The Isoelectric Focusing part will be due:
  • Tuesday People: Thursday, Dec 12th
  • Wednesday People: Friday, Dec 13th

12/10/02:
As it turns out, you can use the prestained (with the different colors) standards for helping figure out the molecular weight (Rm vs log(molecular weight), rather) standard curve. The values in the manual are correct for those, as it turns out - it's the same tube of standards that we were using last year. I will put up a picture ASAP of the colors for each one, and/or you can come by and take a look. In regard to who had which sample in which lane in the isoelectric focusing gels, here's the info I have so far (if you know more, please email me):

• Tuesday Lab:
  1. Standard A?
  2. ?
  3. Jenna + Johanna + Yiannos
  4. ?
  5. Darius + Tomas + Michelle + Nitya
  6. Lynda + Tracey + Dee
  7. ?
  8. Standard G?
• Wednesday Lab:
  1. Emelia's Standards
  2. S + R
  3. P E A
  4. K D F
  5. V K M
  6. E E M
  7. E M J
  8. ?

12/16/02:
In response to people's questions:

• The exam is in Ruth Adams room 001 (the lecture hall).
• I expect to have the electrophoresis/isoelectric focusing lab reports ready to hand back at that time, at least for those turned in by Friday or so... if I am left undisturbed this evening and night!

3/11/03:
Below are the pictures from Tuesday lab; I'll do some work on sharpening them up (sorry about not having done this already, but dissertation research proposal work does have to have a higher priority...):

• jlygel.tif
• kbjhrsgel.tif
• lptmgel.tif
• ecmdgel.tif

3/13/03:
OK, here are the cleaned-up versions (in TIFF, GIF, and JPEG formats):

• • jlygel.tif
  • jlygel.gif
  • jlygel.jpg
• • kbjhrsgel.tif
  • kbjhrsgel.gif
  • kbjhrsgel.jpg
• • lptmgel.tif
  • lptmgel.gif
  • lptmgel.jpg
• • ecmdgel.tif
  • ecmdgel.gif
  • ecmdgel.jpg

4/5/03:
I am in (room 118 in Lipman, usually) and available for a limited amount of helping people with the plasmid lab (I hadn't anticipated being in this evening, but my cold makes it inadvisable for me to do much talking due to a sore throat developing (keep that in mind when coming by for my assistance - I suggest email if at all possible!), so I'm in). See below for how to get in touch with/locate me (making very sure to read over the information on emailing me carefully so you don't send email to an address where it will get mistaken (by a program) for spam... I have taken precautions against this happening for email from Rutgers computers, but other email accounts are another matter entirely). The plasmid lab is due, essentially, anytime before Monday, if you didn't get that info. If people did not get a copy of the 1 Kb DNA Ladder, the standards, it is available at 1kbLadder.png or 1kbLadder.gif on this server. Note that the 1636 Kb band is likely to be brighter than the bands above and below it, although the 1018 band can be easier to locate in some circumstances.

Some things regarding the instructions on page 26-28 on the Report:

1. You do need to give me a circular map (one for each lane - except the standards - and one for the entire plasmid as you've reconstructed it), since there's no realistic way that you can reconstruct the "linear sequence of the insert" (or, rather, the restriction sites in the insert) without it - so don't bother with a seperate linear map, just give me the circular form (all you have to do with the vector part is label it vector and give how many BP it is).
2. I don't advise using semilog graph paper, at least if the relationship between log molecular weight and distances appears to be reasonably linear - human drawing is too inaccurate, although it can be better at accomodating weird curves than a computer is unless the computer is very well programmed.
3. For the instructions in #9, ignore the references to digoxigenin-labelled standards.
4. For #11:
   1. Tell me what the equation is that you used to convert from the blot measurements to the gel measurements (physical measurements on the gel will generally not correspond (in non-relative distances) to measurements on the blot). I don't advise people to try converting to the physical gel measurements, BTW - that's a waste of time; just be sure that you convert your blot measurements to the same set of measurements as you use for the standards. I really don't even care what the physical gel or blot distances were - just the distances on the photographed gel and the distances on the photographed blot.
   2. Tell me the data points you used to get the conversion equation (I have a program that I can use for getting the conversion equation if you tell me the distances to be used for the conversion) - note that I am finding this matters quite a bit, and it is preferable that you use points from both sides of the gel if at all possible (to allow for distortions in how the gel ran, such as one side moving faster than the other).
   3. See about figuring out where the probe bound (not to what "sequence" the probe bound to (you don't have the plasmid's sequence (yet))) in relation to your restriction map - note that you will almost certainly be somewhat unsure on this, especially since we are also - by comparing what showed strongly on the blot, and not on the gel (which corresponds to the restriction fragments that the probe bound strongly to), to what showed strongly on the gel, and not on the blot (which is what the probe did not bind strongly to).
5. The standards are not in kbp (kilo-base-pairs), but in bp (base-pairs) and you should be working in bp; working in kbp is a bit silly with a plasmid of less than 10,000 bp.
6. If your gel picture is reasonably clear, a photocopy with the bands you used outlined is sufficient to fulfill the requirement for a "drawing" - this isn't an art class. The same is true of the blot. (Just turning in the blot, however, is not sufficient, as Dr. Chase states.)

4/6/02:
I'll be in until at least 1 AM or so. Keep in mind that you're working with a log scale - so a minor error, difference in measurement due to fuzziness of the band, etcetera can easily make a big difference in the estimated BPs - as in 800 BPs or so. Another factor is that the smaller bands tend to have a better measurement of their size than do the larger bands (the standard is more reliable and not so blurred together), so the linear band may not be as accurate an estimate of the size of the entire plasmid as the sizes of the fragments put together.

4/8/02:
Unless I hear otherwise from Dr. Chase, the due date is now today, due to the snow yesterday. The one person who got hers in yesterday (plus the other person who hasn't been able to complete it due to her lab partner not making the data available - if it isn't due to illness, he's not going to be happy with the grade he gets) will be getting extra credit. I'm available today, keeping in mind that I'm still congested and can't talk for long without getting a sore throat, but I am feeling somewhat more energetic than I was last week. A few things to note:

• In the description of what is needed for the report that Dr. Chase gives, that description is not in the actual order that you (will almost certainly) need to do the actual procedure in - it's what makes sense as how the final report is set up. The sequence for dealing with the gel and blot pictures I suggest it is as follows:
  1. Measure the distances of the bands on the gel and blot pictures, preferably on as enlarged a picture as you can get, with the zero point being the well for that lane (if possible).
  2. Figure out which bands on the gel and which bands on the blot clearly correspond to each other. Come to me (or do a least-squares equation yourself) to figure out an equation for taking a blot distance and getting out the equivalent gel distance, using said corresponding bands. Note down and include in your final lab report the bands used for constructing the equation as well as the equation itself. Put into the table for the blot values both the original blot measurement and the equivalent gel measurement. The blot measurements are of use in determining where the probe bound (see below) and, if a band showed up on both the gel and the blot, confirming that the band in question seems "real".
  3. Figure out what band on the standards (on the gel) is which, keeping in mind that the 1636 band is typically brightest. Use this bp (not kbp) information (note that 506,517 on the standards is actually 2 bands, one with 506 bp and one with 517 bp - not one band from a 506,517 bp fragment, which would be huge...) and the distances of those standard bands to get the plot that Dr. Chase discusses (except that you only put the standard bands on this plot, not any others) and a log(bp) = slope*distance + intercept equation (y = mx + b, where y is log(bp) and x is distance) - alternatively, you may actually find that if the plot you get is curved, it works better to use a slightly different equation (I'm still examining this... feel free to experiment, as long as what you wind up using is at least as accurate as the "standard" equation and you explain the equation that you use). Plug the other gel band distances, including the blot distances converted into gel distances, into this equation to get out a log(bp) value, then take the antilog to figure out how many bp (not kbp) each band represents.
  4. For each lane except lane 1 and the standards (lane 6), create a miniature plasmid (circular, not linear) drawing, as with a normal plasmid map. If a particular lane is giving you headaches (more so than normal...), go on to another lane and later use the data from the other lanes to figure out the problematic lane(s). If you are getting more than one distance between sites (as in one distance from the gel and one distance from the blot), put down both distances as a range (500-600, for instance).
  5. Put together the plasmid maps for each lane into one large plasmid map of the entire plasmid. There will be some uncertainty, particularly about the exact lengths; report the reasonable range of possible lengths of plasmid between the restriction sites.
  6. Using information from what is visible on the blot but not on the gel and vice-versa (note that most of the standard will not be visible on the blot; neither will the RNA), figure out where on the plasmid (between what restriction sites) the probe hybridized to (or at least hybridized to most strongly).
• Do not try to label anything on the drawing except for RNA. Label things in a column in the table that you make up (you can make up either one table for both the gel and blot values, or two seperate tables for each), as one of:
  • RNA
  • Standard
  • Supercoiled (plasmid)
  • Relaxed (plasmid)
  • Linear (intact plasmid, with only one cut)
  • Restriction Fragment (plasmid with more than one cut, producing at least 2 fragments)
  ("Unknown" or "?" may also be an acceptable category, especially if I can't tell what it is...) While the restriction fragments, like the standard bands, are linear in conformation, it is most useful to treat each of these as a seperate category.
• I am seeing very few cases where the "linear" band is visible in lane 1. Use lane 2 instead to get it. Lane 1 is more useful for figuring out what, in the other lanes, is actually supercoiled (undigested) and relaxed (partially digested/broken), not a restriction fragment (unless a restriction fragment band happened to be in the same place as the supercoiled/relaxed band, which is unfortunately possible).

4/11/03:
I will be in and available this weekend, with the exception of Saturday evening/night. Afternoons and (except for Saturday) evenings/nights are preferable. Note that, in calculating the concentration of DNA, the lab manual has a clarity problem in that if you used 2.5 ul of sample with 2 ml of water in the cuvette, it should be a dilution factor of 800, not 400, at least for figuring out the nucleic acid concentration from the absorbance (but a dilution factor of 400 is correct for the fluorescence measurement). (Don't worry about this on your lab report if you've already turned it in, since I hadn't noticed it until now...) Things seem to have worked despite this, interestingly enough. (BTW, regarding the RNA gels, I will put up previous ones for people to analyze (since their own didn't work) once I get more plasmid lab reports in or someone who has turned their plasmid lab in requests it.)

4/13/03:
The questions:

1. Appropriate concentrations are not of that much importance for the first and third parts of the question - think about how restriction enzymes work, and that they leave complementary sticky ends (and that you can't ligate things back together if the sticky ends aren't complementary). The answer to the second question is simpler than it looks, and can indeed be done in terms of concentration.
2. Page three of the introduction mentions a bacterium whose DNA is being pelleted (to remove the chromosomal DNA, the size of which is what you need to know for the question); assume it is E. coli, even though it doesn't say the species.
3. Don't worry about the Boehringer blocking agent; assume that it's the same thing as sheared (randomly chopped up) salmon sperm DNA. Note that in this case, it isn't a question of "what might you see if you didn't do this?" - it's a question of "what did we see because this (the procedure mentioned in the first part) answer to the first question) didn't work very well", and that Dr. Chase wrote the lab assuming that the blot would only show parts of the plasmid from which the probe DNA was derived. This didn't happen. Think about what happens when randomly-chopped-up pieces of DNA (the sheared salmon sperm DNA) get added to the blot, which has a lot of DNA bound to it, some of which is complementary to - and thus binds tightly to - the probe, and some of which the probe is no more complementary to than it (the DNA on the blot) is to the sheared salmon sperm DNA.
4. Most people seem to get this one. Please let me know if there's anything unclear about it.
5. He's referring to the hybridization procedure, after you've added the probe but before you add the dye. The hybridization we're trying to make stringent is the one between the probe and the DNA on the blot (as per what I said above, we didn't succeed in making it very stringent - this actually wound up helping us, since the blot is more sensitive than the gel for plasmid DNA).

RNA:
None of the RNA gels (at least on Tuesday, and for most people on Wednesday) turned out very well - or, actually, for anything other than the standards, with anything at all usable I'm afraid. (Given the consistency of this, it's pretty clear that this wasn't something you did that caused this (in most cases - some were a bit worse than others). We'll be looking at the procedure for ways to make sure this doesn't happen again - while RNA is notoriously difficult to work with, nobody having usable results on an entire day should not be normal. I can say that it looks like the liquid nitrogen preserved the RNA better than the dry ice; instead of the RNA getting chopped up, we instead seem to have had more problems with too much DNA being left in, as seen by the bright wells.) We are therefore going to provide all of you with a picture of a gel of previous results (to be precise, by Rosa, a past student of Dr. Chase's). This picture is available as RNAGel.tif, RNAGel.jpg, RNAGel.png, and RNAGel.gif (in the appropriate file formats for the names; the .tif one is the highest-quality, but is also the largest-size and is not as portable as the other two formats). Going from left to right, the first lane is the standards (the same ones as you got - see rnastd.jpg if you didn't get a copy); the second lane is RNA from another plant; the third lane is how the cabbage leaves you worked with should have turned out; and the fourth lane is from E. Coli RNA. The (unfortunately faint) black rectangles close to the top, with thin bright lines right below them in some cases, are the wells. Note that you don't need to worry about what the physical distance was on the original gel, just on the picture. You can use the end of the picture (away from the wells) as the "dye front". You should use the 2 bands from the E. coli rRNA (in the rightmost lane) along with the info in the introduction to the lab on prokaryotic rRNA sizes to get a sufficiently accurate standards graph. Dr. Chase may want you to use a curve instead of a line for the standards (I'd say to do this only for the portion of it that is curved, with a computer equation line being used for the rest, or else figure out an equation on SigmaPlot for the curve); however, ask him, not me, especially since I'm not grading this lab.

• Unless you are using semilog graph paper (if for some reason you wish to do this, it's allegedly available from the Cook/Douglass bookstore), you do not need to label the graph in kilobases vs distance, you need to label it in log kb vs distance.
• Ask Dr. Chase how much of the procedure he wants written up (I suspect he will want more than I had asked for in the earlier version of this writeup - sorry!)
• You can use the equation of the line to get the log kb (and convert that into kb) instead of measuring off the graph, provided you look at the equation of the line to make sure it makes sense... at least, you can do this for the portion of the line that isn't a curve, or with an equation for the entire line including the curve.

4/25/03:
Since most people didn't have results for the PCR gel (probably because of too much RNA and plasmid DNA instead of genomic DNA in their samples), we're putting up the one gel that worked, and a PCR try and gel that the Wednesday Lab TA, Maria Cruz, got:

• JLY group from Tuesday:
  • jlysb.pcr.tif
  • jlysb.pcr.jpg
  • jlysb.pcr.gif
• Maria:
  • maria.pcr.tif
  • maria.pcr.jpg
  • maria.pcr.gif

• The bottom lane is a (positive) control (to make sure the PCR machine was working);
• The next two lanes (going up) are from a couple of the groups (H and M, I believe);
• The next three lanes are a 1:10 dilution, a 1:100 dilution, and a 1:1000 dilution, using the D (not A) group as a basis;
• The topmost lane is a 1kb standard ladder (see above);
• The primers used for D were F27 and R1522.

4/29/03:
The sequencing labs are due tomorrow, 4/30/03, by 6:00 PM, in Maria Cruz's mailbox in Lipman Hall. The PCR labs are due next Monday, May 5th, by 6:00 PM, again in Maria Cruz's mailbox in Lipman Hall. There are a couple of changes to the PCR data above; please take a look at that.

4/30/03:
I am afraid I misinformed some people regarding the RNA standard bands. Do not skip over the 1.35 band in the standards; it does indeed appear. The heaviest two bands (7.46 and 9.49) are, however, merged into the band at the top (closest to the wells).

Comments

People frequently say that the lab takes more time than a 2.5 credit course should. 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.

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.

Intro Sheet

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

1. prior lab experience, especially with DNA; and
2. having TAed it before (this will be my 5th or 6th time for the spring 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). (Do not assume that I'll receive voicemail; only use this method if I (or someone else) answers 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, 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; the best thing to do is to simply ask whether I will be available at a given time - people who've had me before can tell you that I am willing (unless other obligations, including my own academics, 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, given the prelabs you are being asked to do, I will not ask for any further quiz-taking.

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

• Good things:
  • asking an intelligent question;
  • giving an intelligent answer on a quiz;
  • giving an amusing answer on a quiz;
  • typing a take-home quiz;
  • typing lab reports, particularly the ones that I grade (which are the gel electrophoresis and isoelectric focusing ones for the Spring Semester);
  • doing significantly more work than other people;
  • giving a good answer to the questions on a lab that I grade;
  • helping someone else in the lab (especially someone not your lab partner);
  • cleaning up messes that you didn't create;
  • doing extra work because of things that aren't your fault;
  • pointing out problems to us (especially if you provide the solution also);
  • for the Spring Semester, choosing to do the Carotenoid lab (generally considered harder but more interesting - there have been exceptions to this, depending on sources of material chosen, however) if most people are choosing to do the Lipid lab
• Bad things:
  • asking a question that makes it apparent that you haven't read over the lab;
  • doing something that makes it apparent you haven't read over the lab (more off for this than the first - if you don't know something, do ask);
  • coming in more than 15 or so minutes late without an adequate excuse (this may also result in your missing a quiz - which you will not be able to make up without a good excuse for coming in that late);
  • leaving a mess that I or Emilia have to clean up (particularly the latter, if she has cause to complain to me);
  • doing significantly less work than your lab partners;
  • doing something that irritates your lab partners (unless you and they come to me and/or Dr. Chase and explain the problem and we decide you're in the right)

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, 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. On lab reports and quizzes, I'll circle the final grade that you'll get for each of them. Such curving does not include extra credit points or points taken off for lateness.

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

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