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http://www.drallensmith.org/teaching/index.html.
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My Teaching: TAing Experimental Biochemistry - 2003-2004 webpage
I TA one of the labs in the course
Experimental Biochemistry
(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:
- any information that I need to get to
students (or would like to make available to students) right now
- any comments about the course that I wish to
make
- 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
http://www.drallensmith.org/teaching/index.old.html.
For the 2002-2003 web-page, see
http://www.drallensmith.org/teaching/index.2002-2003.html.
I am afraid the below is somewhat out of date, at least with regard to the
Intro section and below it.
9/9/03:
We are not requiring prelabs, at least for the Tuesday labs. Nor do I plan
on requiring them for the Tuesday labs next semester.
12/03/03:
Here are most of the SDS and native (activity-stained) gels from the Tuesday
lab. I will post any others I get, for either lab section, with some image
reworking/improvement on them (the below have already been modified); note
that this will need to be on disk (3.5 preferred), not emailed (I don't do
attachments). I will be available from Thursday late afternoon on for help
with the writeups for the SDS-PAGE/Isoelectric focusing lab reports,
including getting the interpretation of the densitometry data out of those
computers (note that if you did it on the laptop, I will need to be notified
prior to 5:00 PM or so on a given day, so I can get the laptop back from the
room it's locked in (I don't have the key).
- johnsds:
- Ankit:
- diane_dan:
- John (antibody-stained blot (membrane)):
(If you have problems viewing these, please let me know - I can translate
them into other file formats than TIF and JPEG.) In regards to assistance on
the SDS-PAGE/IEF lab reports, I can open up the 219 computer room for use of
those computers. (Note that the SigmaPlot version on them is 4.01, which
can't read SigmaPlot 8.x files!) Moreover, I will usually be in until 1:00
AM or so for consultation and (next week) for turning in the lab report
(it's in on a given day provided it's to me before I leave the next morning,
unless it comes in after 8:00 AM the next morning). (Note that turning in
lab reports via email will not work unless they are in plain text format -
as in what Notepad could produce - and since you will need graphs, that
won't work. I don't accept Microsoft Word attachments (I don't use an M$
Windoze computer to read email), and whether or not I even let you know that
it wasn't accepted will depend on how good a mood I'm in... I
really
don't like Microsoft!) Call me
(732-932-9255 x119) to get let into the building after it's locked up, or
throw a stick or something (not a rock!) at my window (the farthest to the
right on the front of Lipman).
12/04/03:
OK, here are the coomassie and activity-stained IEF gels for Tuesday and
Wednesday:
- Tuesday coomassie IEF gel; a rather torn-up
gel; see below for data measured by Dr.
Chase when it was more usable; its top and bottom should be along
where the electrodes were:
Note that I have chopped off the right-side standards, which are
actually the ones that Dr. Chase measured, because they got majorly
messed up when Emilia touched the gel with a paper towel (that's the
reason for the shaded area).
- Tuesday activity IEF gel:
- Wednesday Coomassie IEF gel:
- Wednesday Activity IEF gel; only the top of this
one is along where the electrodes were, I'm afraid, but it should be
reasonably obvious how to convert measurements from one scale to the
other:
Here's the data that Dr. Chase measured for the
positions on the Tuesday Coomassie Blue gel, measured from the top of the
gel, using the standards on the right side of the gel:
- Phycocyanin:
- Beta-lactoglobulin B:
- D-amino-acid oxidase (major band):
- Bovine carbonic anhydrase:
- D-amino-acid oxidase (heaviest of minor bands):
- Human carbonic anhydrase:
- Myoglobin:
- Hemoglobin A:
- Hemoglobin C:
- Lentil lectin:
- Lentil lectin:
- Cytochrome C:
Plot the cm vs pI and see if you get a reasonably smooth curve, then
interpolate to get the pIs of the major and minor D-amino-acid oxidase bands
(plus the others - Dr. Chase only measured the ones closest to
the standards). (You can actually either use these measurements,
from the Tuesday coomassie gel, plus your own measurements off of that gel's
photo (remember to convert the scaling!) and the Tuesday activity gel,
or use the Wednesday gel and the above info to figure out which
standards were which from that gel and do your own measurements (while
you'll need to do all your own measurements on the Wednesday gels, you will
be working with gels that are rather easier to measure!).)
12/05/03:
Due to the snow, I'm going to ask Dr. Chase if we can delay the due date of
the SDS-PAGE/IEF lab by a couple of days, until Thursday/Friday (for
Tuesday/Wednesday labs). [They are delayed; see below
for more information.] I am here in Lipman, and probably will be until
Sunday morning/afternoon. I will probably be in my office (118) most of that
time, but will crash on a couch to sleep at some point!
12/07/03:
Here are two more gels:
- Coomassie:
- Native activity:
The non-prestained (single color, only showed up after staining with
coomassie blue) standards are the ones given in the lab manual; the carbonic
anhydrase band tends to be the heaviest, with two bands moving further than
it and two bands not as far (no phosphorylase b protein - she did use one
very large protein, but it doesn't seem to have shown up on any of the gels
I've seen so far) - that's on page 17 of the lab manual. For the prestained
standards, see below. I will be in
this evening quite late (until in the morning, really) - call or tap on my
window to get in (actually, I suggest calling in any case, to make sure I
haven't gone upstairs to take a nap). Incidentally, regarding the gels/blots
and the mention of 'drawings' - you do not have to do full drawings
of any of them, if you have a picture and
you have drawn on it where the bands are that you measured. This isn't an
art class...
12/07/08:
I'm going home (it's about 4:20 AM) and should be back sometime between 1
and 4 PM.
Well, I overslept by about an hour... sorry about that, but I couldn't get
to sleep until after 9 AM! I am here now and available for consultation; I
should be available also sometime tomorrow afternoon (4-5 PM is likely).
The labs are now due Thursday (for the Tuesday lab) and
Friday (for the Wednesday lab), but if they're in later than Friday
(including over the weekend), you will not be getting them
back at the final and you may be getting a T grade. I will
not be available for consultation after Friday - I'll be
too busy grading so as to try to get back the reports at the exam, and
hopefully be able to put up (on here) some comments on them that may be
helpful for the exam (see below for my comments on
the exam, BTW).
I have put up above some additional versions of the
gels I've scanned so far, namely in JPEG/JPG format as well as TIFF, since
some browsers apparently have problems with TIFF. (To print out the gel
pictures, bring them up in a web browser - a word processor probably won't
do it!)
The prestained standards (multicolor) are as per
the ones on Emilia's door, the Kalideoscope standards, namely the ones with
the red "X" below them:
- Myosin:
- B-galactosidase:
- BSA:
- Carbonic Anhydrase:
- Soybean Trypsin Inhibitor:
- Lysozyme:
- Aprotinin:
Here also is one more (native activity-stained) gel:
12/10/03:
When Dr. Chase says to compare the bands you get for the antibody and the
bands you get for the coomassie (or, for the native gel, the activity
stain), and those you get for measurements via hand and those via the
densitometer, the best way I know of to do this is to calculate the Rm/Rfs
and corresponding log molecular weights and molecular weights. (Indeed, I
don't know how you would do it otherwise - that would be what I
would do in doing research myself - but if you can come up with some other
way, fine.) Also, in determining what the D-amino-acid oxidase band is, and
its molecular weight, you should also be calculating the molecular weight of
the other bands (for the SDS gels), partially since given a log scale what
looks similar to the eye can be deceptive (again, if it's obvious which one
is the D-amino-acid oxidase band, then you are not strictly required to put
the info down for the other bands, but I do prefer having it).
In terms of how much writeup of the procedure you should put down, I am not
interested in reading 44 copies of the same procedure. Put down any amounts
you used, any measurements you made, any way in which the procedure deviated
from that in the lab manual, and anything you noticed that was different
from what the lab manual said to expect.
12/12/03:
If you have already turned your lab in and followed the
directions above and in the lab manual, don't worry about the below, except
for future reference (e.g., potentially for your future lab work, or even
for the exam).
- Scheduling
- I will be in and available for questions until midnight. After
that, I will be grading and not available for questions (or for
turning reports into, or for getting let into the building). You
can turn reports in over the weekend (before Monday) and they
will be counted as being in on Friday - if you can get them into my
box (if someone else lets you in or you have a key to the
building). I am unlikely to have them graded by the exam if they're
in after midnight, however - I'm worried already about having the
ones that get turned in on time back at the exam, especially since I
seem to be coming down with an infection of some sort!
- Graphing
- Please do not put points for your D-amino-acid-oxidase
band on your molecular weight graphs, or for your isoelectric
focusing graph, if you are using the equation of the line
to figure out log(molecular weight) or pI from the distance/Rm/Rf. The
lines should only contain points for your standards. (If you've
already turned in the lab, don't worry about it.) The only time you
would have a marking for a non-standard on such a graph is if you're
using the method of drawing a line on the graph between two of the
standard values in order to manually figure out the log(molecular
weight) or pI from the distance/Rm/Rf.
In regard to what goes on the X axis and what goes on the Y axis, I
don't care. I advise people to put the distance/Rm/Rf on
the X axis, so that once you get a y=mx+b equation, you can just
plug in distances/Rm/Rf to get a log(molecular weight) or pI.
- Isoelectric Focusing
- Again, you can use either the tuesday or the wednesday
lab gels for this. If you use the wednesday lab gels, then you do
all your own measurements, including of the standards and of the one
or two bands on the activity gel - and use the standards
above for the pI values but not the
cm. If you use the tuesday lab info, you will be measuring all 5 of
the bands on the activity gel, using the main one to get a
scaling/conversion factor between the gel picture and Dr. Chase's
measurements, and finding the pI of the rest using the graph.
For the tuesday lab data, you will get a pretty broken-up
curve. You will need, as Dr. Chase wrote, to interpolate to find the
pIs - use more than one line. You may or may not need to do this for
the wednesday lab data - it partially depends on which bands one
identifies as being which standards.
- Standards
- I am generally seeing the (pre)stained standards as being less
accurate than the unstained standards. Do try graphing the
(pre)stained stained standards, but the molecular weight values from
the unstained standards generally are more accurate.
I will be TAing the Tuesday lab again next term, incidentally - some people
have asked about this...
12/18/03:
As it wound up, the only labs with things that people should have been told
anything about prior to the exam were unfortunately turned in after Friday,
so I wasn't able to do any feedback from them. (The lower the molecular
weight, the higher the Rm/Rf, people...) I have added a bit to my comments
below.
3/2/04:
- Carotenoid labs:
- The carotenoid lab is now due one day later (and may wind up being further
extended, but that's up to Dr. Chase). You should pick up from myself, Emilia,
Kyle, or Dr. Chase:
- The spectra on the most important of the peaks from each of your
samples (this will need to be done on the HPLC computer)
- The full wavelength info (as in for wavelengths other than 350) for
each of your standards (again, this will need to be done on the HPLC
computer)
- The full wavelength info on the various standards in use (as in
for wavelengths other than 475 (and 350)) - this is in Emilia's room
You may find the site
http://carotenoidsociety.org (and
the site http://lipidbank.jp) of use.
Other than that, Dr. Chase is the one to ask on carotenoids (although I can
help a bit on some aspects, such as the K and K-prime stuff). (He says, BTW,
that you only really need to identify the most significant 4 or so carotenoids
in each fraction, starting from the right-hand side of the HPLC,
although more if you can manage it would be of interest. Anything
with a maximum absorbance less than 5 uA is probably not there, as
is anything below 4 minutes Rf time (certainly anything below 3 minutes).)
- Lipid Labs:
- I am available to help with interpretation of your lipid lab results. You
will probably need to compare info with another group in order to interpret
all of the GC results, such as for peaks that the computer mis-identified
or failed to identify (probably due to too tight tolerances for what it will
accept, or that the peak was too wide for it to get an accurate retention time).
I suggest taking a look at http://lipidbank.jp
for information on the common names of lipids (don't worry too much if you can't
find a common name for stuff that isn't a huge portion of your samples).
3/2/04:
Agarose gel pictures, in 3 different formats:
- MIK (Plasmid H), Tuesday:
- ERA_Mar2, Tuesday:
- Version 1:
- Version 2 (edited to remove most of the dye blackness):
- HDIN, Tuesday:
- Andoria/Nipa/Liz, Tuesday:
- Dee/Sharon/Aaadya, Wednesday (Plasmid D):
- Pawan (plasmid H), Wednesday:
Standards:
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.
I will put up blot pictures if given them; please do give copies to me so
that I can do so. Here are the ones that I have been given so far:
- Tuesday, Andoria/Nipa/Liz:
- Tuesday, ENJ:
- Tuesday, Magda et al (Plasmid H):
Do NOT yank on the side door(s) to try to get them to pop
open. Doing so damages the doors. If I catch anyone doing so, I will see to
it that they are:
- Flunked from the course;
- Brought up on charges for damaging Rutgers property; or
- Both.
3/17/04:
Yes, I'm here over break, and available for some limited assistance
on the plasmid labs - I will be available for a lot more assistance after
the break, but this week, I need to catch up on sleep/laundry/etcetera! I
suggest emailing me to schedule something; I am in this morning but probably
not this afternoon, for instance.
3/21/04:
I am not feeling very well (congestion (resulting in earaches), sore
muscles, etcetera), but will endeavour to be as available as possible for
help on the plasmid lab, which I am grading. A few things:
- You don't have to do a drawing if you have a photograph or
computer-scanned/whatever image, provided you mark on that
photograph/printout exactly where you are measuring the bands
to. This goes for both the gel and the blot.
- I have no desire whatsoever to read over
50 (or whatever) copies of the lab manual. What I want on the
procedure is:
- Any deviations from the procedure in the lab manual
- Anything that went wrong
- Any observations that you made, including all numeric
measurements
- All amounts of stuff that you used (and any other
similar numbers, like the voltage used to run the gel) if it
wasn't the same as given in the lab manual
Other than the above, just reference the procedure in the lab
manual. (If you've already written up the procedure in full, don't
worry about it; just stick it in, after making sure that all of the
above are in it and reasonably easy for me to locate.)
- Typing everything except calculations will be appreciated. If it
isn't typed, it must be legible - if you have any
uncertainty as to whether something is legible, and it isn't
calculations, type it. If it is calculations, ask me whether I can
read them.
3/23/04:
Here are another couple of blot/membrane pictures:
- SJV, Tuesday lab
- HDIN, Tuesday lab
A few other things:
- Plasmid lab:
- Don't worry about figuring out what is
supercoiled/linear/fragment until when we're/you're working
on doing the restriction maps. You can't tell what is
supercoiled and what is (or is also) a fragment that runs
about where the supercoiled does until then.
- Don't worry about the physical gel/blot distances - they
will probably not coincide very well, unless your blot
stretched/whatever less than most I've seen. You instead
will need to work out an equation to convert from blot
distances to equivalent gel distances - use any plotting
program, or I have a program that I can stick your
information into and get out an equation + some idea of
which points may need to be excluded (you should only use
the equivalent points from blot and gel that you are pretty
sure are equivalent).
- RNA Lab:
- Unless the procedure says otherwise, keep things cold!
- Use gloves, and change your gloves whenever you are
likely to have touched something that hasn't been cleaned
of RNAses (e.g., your forehead or nose!).
- We are likely to be using thicker gels (and being sure
to use the thicker-toothed side of the combs) than is in the
lab manual, so as to be able to fit an adequate volume of
sample into the gel.
- We are hopefully using liquid nitrogen, which should
increase our success rate as compared to the past (when we
were using dry ice and sometimes had only one or two groups
getting results...)
I am probably mostly going to be around in the evenings, although not for
that much longer tonight (only an hour or so longer). Hopefully, I'll be
feeling better by this weekend or so...
3/26/04:
The Plasmid lab is now due (for both lab days) on Friday, April 2nd (and
will be counted as in on Friday if you can get it to someone who can sign it
over the weekend; note that I cannot read attachments and may not receive
them, so don't try sending labs to me as a Microsoft Word file or similar).
I am feeling slightly better, and should be able to be here tonight
until midnight or so, then on Saturday again in the afternoon/evening.
3/27/04:
Please let me know what letter of plasmid your group had if a picture from
your gel or blot/membrane is on this webpage. I will be here until midnight
or so (let me know if you're planning on coming later than 9 or 10, or I
might leave earlier than that; I'm still needing more sleep than normal...),
and again on Sunday.
3/29/04:
I'll be here tonight until around midnight; I may be busy around 8:00 or so,
however. Here are a few more blot/gel images:
- Diane/Dan, Tuesday (Plasmid D); I suspect we'll try taking a
picture of the blot using the camera instead of a scanner:
- Michael Neu et al (Plasmid F), Wednesday:
- Gel:
- Blot (negated; the initials were at the bottom
right-hand corner):
- Renee/Aaron Blot (Plasmid E), Wednesday:
3/30/04:
Here are some RNA gels:
- Diane and Dan, Tuesday:
- JSV, Tuesday:
- ENJ, Tuesday:
And here are the RNA standards:
4/9/04:
I have still not received any Wednesday RNA gel pictures - come in and give
them to me, even if you think you don't have anything. If necessary, if I
have taken a look at your gel (including on Tuesday during the lab)
and concluded it wasn't usable, you may use the below, 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:
(in the appropriate file format for the names; the .tif one is the
highest-quality, but is also the largest-size and is not as portable as some
of the other formats). Going from left to right, the first lane is the
standards (see above); 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 if you
use this gel, you will need to do either a by-hand curve fitting for the
standards, or figure out a way to get an accurate curve from them on a
computer (for the latter, and possibly the former, you will need to use the
E. coli rRNA sizes as additional standard bands). You may therefore
wish to use another gel.
4/12/04:
The due date for the RNA lab will be pushed back - we are not sure
by exactly how much as yet, however (possibly until this Friday?). (Dr. Chase
is grading it, BTW.) Peter Anderson is currently working on the computer
connected to the Photodyne (gel camera) - that hard drive may have some
(more-)usable gel pictures on it.
Update: Sigh... the hard drive on the computer connected to the gel camera
is a paperweight (defunct). Use either:
- one of the Tuesday gels; or
- the one that Rosa did.
Either is fine, as long as you interpret it properly (as well as possible).
The lab is (currently) due Friday.
4/14/04:
- RNA: I've been told that some people are having problems finding
some of the RNA gel info on here (perhaps due to link problems,
which have been corrected - sorry about those!).
A possible equation for fitting to Rosa's gel's standards,
including the E. coli rRNA:
log(kbp) = a + b*(cm) + (c/((cm)^d))
In the above, b should be negative, c should be positive, and d
should be greater than 1. You will probably need to use SigmaPlot to
do this curve-fitting; I doubt Kalideograph is capable of it, and
I'm pretty sure Excel isn't. Other equations are OK if they fit the
line reasonably well (as in better than a linear equation), as is
manually plotting the curve on semilog paper.
- Sequencing: I am grading this lab. The lab report is not
very difficult, except for the questions on the sequencing lab. Note
that if I put together a sequencing question for the final exam, as
I have done in past years, you will need to understand how dideoxy
nucleotide sequencing works
and be able to think about it in order to answer
said question. If you have problems with answering the sequencing
lab questions (unless said problems are due to difficulties with
English or different people's writing/reading styles), then you will
have problems with this question on the final if it's on there.
4/19/04:
I am discovering that other equations than the above do not actually fit
Rosa's gel very well (as in well enough for bands to be clearly identifiable
as 18S, 5.8S, 5S, or whatever)... and that some of the RNA gels from Tuesday
give sufficiently curved lines that it may be best (at least to make
Dr. Chase happy, since he's the one grading it) to use the above equation to
fit them also. Note also that it appears that, despite what it says on page
2 of the lab manual, plants have 25S rRNA, not 28S, which
has a smaller size. Since the lab manual got this wrong, you should not be
penalized for saying 28S, but you may find it easier to fit the bands using
25S instead of 28S (25S is smaller).
The Sequencing lab is now due Monday, April 26 (4/26/04).
4/20/04:
I am pleased to report that every lab group on Tuesday got
something (even if only a faint band) in their 1/1000 dilution for PCR, and
all got strongly present bands in all lesser dilutions (and, in a few cases,
even in the 1/1000 dilution). Both standards also worked. The first standard
is the same as the
1KB ladder for the plasmid lab; the
second standard is shown below:
This is an Omega-X174 DNA phage cut with HaeIII, BTW; we are pleased that it
worked (mostly) despite the marker being several years old (one reason we
ran it along with another marker). The pictures that I have so far are as
follows, including which PCR primers they used:
- MIK (1522R + 702F):
- combined (recommended):
- high-exposure:
- low-exposure:
- ENJ and Dan (Diane was (unavoidably) absent); the ENJ bands
(primers 1522R + 357F) are on the left-hand side, and Dan's (primers
1220R + 704F) on the right-hand side:
- combined (recommended):
- high-exposure:
- low-exposure:
- INHD (1522R + 704F):
- combined (recommended):
- high-exposure:
- low-exposure:
- SJV (1220R + 357F):
- combined (recommended):
- high-exposure:
- low-exposure:
- Andoria/Nipa/Liz (1220R + 704F):
- combined (recommended):
- high-exposure:
- low-exposure:
4/21/04:
(The sequencing lab is now due Tuesday/Wednesday, depending on class period,
since I won't be able to start working on the plasmid labs until at
least Thursday of this week.) PCRs on Wednesday likewise worked out well, I
am pleased to say. One group did have the problem of first not putting the
EtBR in the gel, then not putting enough of it in the
samples. OTOH, while their gel is not usable (due to the lower standards -
in the region of the PCR'd DNA - not being visible), it does show the value
of putting (enough) EtBR in the samples/standards instead of in the
gel. EtBR produces a fog in the end of the gel near the wells, which is
rather of a headache to compensate for afterward (as I've been learning with
my attempts at processing the gel pictures); try comparing
their gel to those of others, particularly
in the versions prior to combining (during which I removed as much of the
fog as I was able to do). The groups (with primer info if I have it) and
their gels:
- Aaron and Renee (1522R + 357F):
- combined (recommended; I was assisted in doing this one
by a computer program contributed by Michael Neu, BTW -
thanks!):
- high-exposure:
- low-exposure:
- Sharon/Aadya/Dee (1220R + 704F) and Phil+John (1522R + 704F):
- combined (recommended):
- high-exposure:
- low-exposure:
- SPE (1325R + 704F):
- combined (recommended):
- high-exposure:
- low-exposure:
- VNB (primers 1220R + 704F; for comparison with
others, mainly):
- high-exposure:
- low-exposure:
- combined:
4/22/04:
Kyle says the PCR lab is now due 2 weeks from the final lab (in other words,
May 4th and 5th for Tuesday and Wednesday, respectively). For analyzing the
PCR lab bands, we have noticed that the older of the two standards did not
come out completely matching how it is
supposed to look. Use the 1KB standards to
create an initial standards graph, determine the weights of the older
standards using that graph, and then use whichever ones of those match how
it is supposed to look to help (along with the 1KB ladder) in creating a new
standards graph with more points (especially helpful if the 1KB ladder lacks
(reliable) bands around where your PCR products show up). (It is likely that
the differences are due to some of the standard fragments (restriction
fragments of a bacteriophage's DNA) coming back together over time. When we
next use this standard, we will heat it immediately prior to use to denature
any such binding of standards together.)
4/23/04:
When the PCR lab says (on page 8) "Assuming the PCR worked perfectly, making
225 [2^25] copies", read instead "Assuming the PCR worked
perfectly, making 235 [2^35] copies", since we ran the PCR
machine for 35 cycles, not 25.
4/24/04:
In regard to the sequencing lab:
- What I will want for the procedural discussion is the same sort
of thing as for the plasmid lab - see
above. Do comment if you have any idea
(other than that the kits we used aren't very good) on why your
sequence didn't work, if it didn't work, or why it did work, if it
did work. (If you have a spectrum for your sample for sequencing,
that may be one place to look at for this.)
- For BLASTX, if your Query sequence in the
alignments came out with a bunch of Xes (unless these are due to Ns
in your nucleotide sequence), try rerunning the BLASTX search with the
Low complexity box (by the Choose filter
link) unchecked. Similarly, for BLASTN, if your
Query sequence in the alignments came out with a bunch
of Xes, try rerunning the BLASTN search with the
Mask for lookup table only box checked. This
will make your figures for percent identity for each of the
alignments more meaningful and help you in telling where your query
sequence was the same as a sequence in GenBank, and where it wasn't
(e.g., due to sequencing errors).
- For determining when your sequence started and stopped being
readable, there are two considerations:
- Where does your sequence start having clear/sharp peaks
in the Chromas display, with no Ns in the sequence, and
where does this stop being the case?
- Where does your sequence start being identical to (one
or more) sequences in GenBank (preferably the one from
Dr. Zylstra - with his name in the AUTHORS
section of various records, or his name otherwise
mentioned), and where does it stop being identical to said
sequences? This is done using the alignments from the BLASTN
search.
- You are not required to try fitting your sequence together with
the other sequences, given that most of the sequences were not
usable.
- In answering the discussion part about what sequences in Genbank
are homologous (which is the most important part of the discussion):
- Tell me about what sequences you find that are
identical (or essentially identical - as in identical except
where your sequence had sequencing problems), especially the
one published by Dr. Zylstra.
- Look at the nucleotide sequences and what region of the
gene your sequence matches up to (the numbers (###...###) by
the CDS on the left - see which of those ranges
contains where your sequence starts and stops matching (by
the alignment), and what that protein is).
- Look at those sequences and figure out what the function
of the protein coded for by the gene is. (For BLASTX, I
suggest looking at the ones containing a '|sp|', e.g.,
gi|2495000|sp|Q63931|CCKR_CAVPO - they are most
likely to give good information. For these
SWISS-PROT records, the sections to look at (as
well as the name/title, which is important in all records)
are the Keywords and Comments.)
You may need to use the link from the numbers by
MEDLINE/PUBMED in order to look at
the papers associated with a sequence to figure this out. If
so, cite the paper in your discussion.
- Then look at the other sequences, from organisms other
than the one your sequence (or, rather, the sequences
identical to your sequence) is from, for ones that are very
similar in sequence (have a low Expect and a
high (above 65%) percent identity) and have the same
function. (Again, you may need to use the link from the
numbers by MEDLINE/PUBMED in order
to look at the papers associated with a sequence to figure
this out. If so, cite the paper in your discussion.)
- You need to include copies of the initial pages of the
BLASTN and BLASTX searches (before the section with the
alignments), plus the alignments for the sequences identical
with your sequence or which are homologous to your
sequence. You may also include some of the records for your
sequence, as long as it isn't too many pages (not more than
a couple per link, please), or the abstracts or other
significant sections (not the full paper!) for papers you use.
- Question 1 is concerning dideoxy sequencing, not Maxim-Gilbert.
I am intending to be here until 1 or 2 AM today, and again tomorrow (Sunday)
from noon until 1 or 2 AM again.
Here are Michael and Barbara's PCR gel pictures:
- combined (recommended):
- high-exposure:
- low-exposure:
4/29/04:
A couple of things:
- On question 1 in the PCR lab, it is supposing that you have a
PCR reaction that will reliably amplify up one molecule, if it is
present, and this is happening for 4 out of every 10 samples (and
the samples are 2.5 microliters in size).
- I will be available for help to some degree, especially on the
sequencing lab, until 1 or 2 AM for today (Thursday) and maybe
Friday. I will be occupied grading plasmid labs most of the weekend,
but can be asked quick questions - via email is preferable.
5/1/04:
I got less than 4 hours of sleep last night. I'm going home and going to
bed. I will hopefully not be back in before noon or so Sunday...
5/2/04:
I am in, but should not be disturbed if at all possible. I am
hoping that I can grade the plasmid labs that were in on time by
Monday, but this won't be possible if too many people disturb me. (Note that
disturbing me unnecessarily is going to cause me to be in a bad mood when I
grade your lab report!)
5/3/04:
I have the following people's plasmid labs graded (and you should be able to
pick them up, although it's possible I may need to borrow them back if I
discover some problem with the grading scheme I made up):
- Aaron R
- Andoria T
- Brandy H
- Daniel W
- Diane B
- Elizabeth H
- Hussain D
- Irene W
- Kathryn A
- Magdalena P
- Pawan B
- Shilpa B
- Victoria P
So far as I know, these are all but one of the ones that were in on time,
although some that were not in on time will not be counted as being late due
to legitimate excuses. (Kyle has the lipid labs that were gotten to him for
regrading ready to hand back, BTW.) I will probably be in sometime late this
afternoon or early this evening; I am writing this at approximately 6:15
AM.
5/5/04:
I have some more plasmid labs graded, and hope to have all of them done by
tomorrow morning (or, really, later this morning; it is currently 12:20 AM
on 5/6/04). FYI from Dr. Chase:
The Experimental Biochemistry exam is Monday, May 10, 8-11 AM, in
Heldrich 106 (that's the DC Chemistry building, officially renamed,
since Ms. Heldrich gave $1 million to renovate it, but no one pays
attention).
5/6/04:
The following plasmid labs are graded and can be picked up (this list
includes the one above):
- Aadya S
- Aaron R
- Adenrele A
- Andoria T
- Barbara B
- Brandy H
- Daniel W
- Diane B
- Earnest A
- Elizabeth H
- Hussain D
- Ifeoma N
- Irene W
- Joseth A
- Kathryn A
- Magdalena P
- Michael N
- Pawan B
- Phil C
- Sandi M
- Sharon S
- Shilpa B
- Victoria P
- Vriendra R
They are currently in my office (118); after I leave today (after the
General Biochem exam), they will be with Dr. Chase in his office.
5/7/04:
All but one lab group's plasmid labs are graded. Currently, the mean is
95.5; the median is 97; the lowest is 78; and the highest is 108. (These
numbers include extra credit bonuses and subtractions for lateness.) I have
started on the sequencing labs but do not have any ready to hand back as yet
(I have to grade enough to make sure the grading scheme I put together will
work, and have to have them around in case I need to go back and make
changes to the grading scheme).
5/9/04 (3:00 AM):
All sequencing labs that were turned in to me on time have been graded (plus
one lab turned in 4/28/04 but excused):
- Aadya S
- Aaron R
- Adenrele A
- Andoria T
- Barbara B
- Brandy H
- Daniel W
- Diane B
- Earnest A
- Hussain D
- Ifeoma N
- Irene W
- Joseth A
- Kathryn A
- Magdalena P
- Michael N
- Pawan B
- Phil C
- Sharon S
- Shilpa B
- Victoria P
- Virendra R
You can pick them up from my office (they're on top of the green computer),
if I'm in or someone with a key is in. If you have turned your (plasmid or
sequencing) lab in, you can email me for answers to the questions for
purposes of studying for the exam. I will probably be coming in this
evening, judging by my current sleep cycles, and will try to email everyone
who asks ASAP. Kyle has some PCR labs graded, and if yours is not
among them but you have turned the PCR lab in, you can email him for a copy
of a well-done lab report to study by. A couple of comments on the plasmid
labs:
- Please be sure to distinguish between kb and bp! Note that
a 1kb ladder is called that because about half the
bands are above 1kb and the other half are below 1kb, not because it
is (usually) measured in kb (if you measure it in kb, it has a lot
of values below 1). A number of people kept putting kb in various
places instead of bp
without dividing the bp from the ladder by 1000.
Using kb instead of bp is fine (if weird, for the
size of plasmid we're working with)... as long as you are consistent
and convert bp measurements into kb measurements.
- A restriction enzyme cutting twice typically produces a line of
bands across a gel in each lane for which that enzyme was
used.
- We did a blot with a probe for a reason (actually, more than one
reason). One reason is that it binds to one specific part of the
plasmid, allowing us to track that part. Another reason is that
blots can be more sensitive than gels, if the probe winds up
not being as specific as it should be. You were supposed to
figure out (or conclude, and show why, that it wasn't doable) where
the probe bound (not what sequence the probe had - that's a mistake
in the lab manual). (Due to said mistake in the lab manual, and that
the question regarding the probe is not particularly conspicuous, if
people didn't answer this, I reduced it from the original 10 points
to 5 points worth, with their grade being out of 95 instead of 100.)
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
http://aesop.rutgers.edu/~dbm/tedchase.html
for old exams with answers (at the bottom of the page). 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 win
(yet).)
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; I can personally recommend Dr. William Sofer - Bill Sofer -
as a teacher, although like everyone else he's human and does have limits to
his patience, which are sometimes reached around exam times for
Genetics/Molecular Genetics if 300 people are bombarding him with requests
for grade changes...), or some equivalent, be prerequisites or corequisites
for the second semester of Experimental Biochemistry - and may suggest this
also for the second semester of General Biochemistry - in light of the many
people coming to me needing help on what I, as a geneticist, would consider
very basic aspects of the plasmid, RNA, sequencing, and PCR labs. While this
would take too much wrangling to get through for next year, he is planning
on adding a strong recommendation for such a course to the description of
Experimental Biochemistry in the course catalog.
I am willing to spend quite a bit of time giving assistance, as you can see
from the scheduling comments above. Some (as
expressed in one anonymously-made comment) may be concerned about whether or
not the people I work with are doing enough on their own:
- When I work with people, I want them to do it
right. This has been known to result in doing somewhat more
work than the lab manual may technically call for (although this
also means that, at least if I'm grading it, the person can
get more extra credit if it's done right).
- Learning styles are quite variable. Sometimes, it takes telling
people things several different ways for them to get it. Sometimes,
it takes leading them through how to do something before they
understand it.
- If someone is asking questions instead of thinking (instead of
asking questions to check their thinking, or because of a
lack of clarity in what appears to be required or in how the
procedure is supposed to work, or because they've struggled with
trying to understand something but, perhaps because of the different
learning styles I just mentioned, just can't quite get it), then:
- This won't help the person on the exam - it'll hurt
them.
- People who don't understand what they're doing and are
just following directions tend to:
- get things wrong and either get points taken off
or have to go back and correct the errors, thus
doing more work; and/or
- not do a very good job of discussing their
results and answering the questions, thus getting
points off there - and, at least
if someone's in my lab section and it's a lab report
I'm grading, then how well you discuss your results
and answer the questions are a factor in your
subjective grade.
- I've gotten pretty good at not showing it, but I do have
a temper. Failing to think is an excellent
way to get me irritated.
Please do not let the above make you less likely
to seek help from me when you need it. While I am indeed irritated
when people fail to think, I find it just as irritating when people
fail to ask for help when they need it and I then have to deal with
the results, either in grading or in a laboratory procedure.
Tuesday Lab
My background: My primary background is in biology, specifically
molecular genetics. I am mainly qualified for this lab due to:
- prior lab experience, especially with DNA; and
- having TAed it before (this will be my 5th or 6th 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). (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 and
my need for sleep, conflict) to work with people at quite odd times and/or
for very long hours.
Quizzes: My quizzes have as their primary purpose encouraging you to
have read over the lab before you come in and making sure that you
know, in particular, safety-related information. I do not expect you
to have memorized all of it; my own memory isn't that good, and I will
generally consult the lab manual before answering questions - but I
will have read over the lab. I expect you to know in general terms
what we are supposed to be doing that day and about any safety
precautions that you need to take, particularly those which can affect
other people. I may - I generally don't unless I get inspired or feel
that you aren't reading over things - give you a take-home,
open-library quiz (or at least a question or two, if not a full quiz)
that is for the next week's lab, again mainly to encourage you to read
it over. Except for take-home quizzes and safety quizzes, I will not ask for
any further quiz-taking.
Subjective Grade: How I do the subjective grade is to note down when
you do something good, and when you do something bad. I will fix a
particular starting grade, and doing something bad will decrease your
subjective grade below this; doing something good will increase it
above this. The starting grade and amount up/down will be determined
by whatever gives a final mean of 85 and a high of 100. Examples of
good and bad things:
- Good things:
- asking an intelligent question;
- giving an intelligent answer on a quiz;
- giving an amusing answer on a quiz;
- typing a take-home quiz;
- typing lab reports, particularly the ones that I grade (which
are the gel electrophoresis and isoelectric focusing ones for the
Fall Semester);
- doing significantly more work than other people;
- giving a good answer to the questions on a lab that I grade;
- helping someone else in the lab (especially someone not your lab
partner);
- cleaning up messes that you didn't create;
- doing extra work because of things that aren't your fault;
- pointing out problems to us (especially if you provide the
solution also);
- for the Spring Semester, choosing to do the Carotenoid lab
(generally considered harder but more interesting - there have been
exceptions to this, depending on sources of material chosen,
however; I recommend nuts as a source) if most people are
choosing to do the Lipid lab - or vice-versa, if that happens
- Bad things:
- asking a question that makes it apparent that you haven't
read over the lab (having read over it and not understanding it
due to writing style, lack of clarity, or whatever - different
people understand different ways of putting things, after all -
is not, however, a problem at all, and if it leads me to make
suggestions to Dr. Chase as to how the lab manual can be
improved, can get you an increased subjective grade);
- doing something that makes it apparent you haven't read over the
lab (more off for this than the first - if you don't know
something, do ask);
- coming in more than 15 or so minutes late without an
adequate excuse (this may also result in your missing a quiz -
which you will not be able to make up without a good
excuse for coming in that late);
- leaving a mess that I or Emilia have to clean up
(particularly the latter, if she has cause to complain to me);
- doing significantly less work than your lab partners;
- doing something that irritates your lab partners (unless you
and they come to me and/or Dr. Chase and explain the problem and we
decide you're in the right)
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.
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