The exam will be cumulative, since we have been improving our skills throughout the semester, but more emphasis will be placed on the material since the midterm. Format will be similar to the midterm, with an emphasis on solving problems. Problems will probably be different than the ones we did in class, but all can be solved using the techniques and principles we have learned in class.
Review the Midterm Study Guide and the main readings from last semester (restriction digests, competency, transformation, etc.). Make sure you are familiar with these important concepts. Also pay careful attention to the proper controls you need for each of the main procedures in Molecular Biology.
Read through the section on "plasmid recombination and identification", pp. 125-166. Understand the conformations of uncut plasmid DNA outlined in Lab 6.
The main new techniques we have covered since the midterm are:
Our Poster Project this semester was a sequencing experiment. Review our Poster Project, including the protocols associated with our project. Know why we did each of the different steps and what we wanted to accomplish, both for each step and our overall goal.
Labs #18-21 (cloning by PCR) also have some useful information on PCR. This series of experiments uses the cloning vector pBLU. You should be familiar with the features of this vector and other modern cloning vectors.
We did not get
the opportunity to discuss replica (differential) plating, and we will
not
test on this.
PCR (lab #22 and poster
project)
Understand how PCR and thermal
cycling works. What are the components of a PCR reaction? What is in
the
Master Mix? How does setting up a PCR reaction differ from a
restriction
digest? What is the difference between the forward (upstream) and
reverse (downstream) primers? Know the main files used in PCR and the
function of each of these
files. See the Pre lab Exercise for step cycle, soak, etc. What PCR
steps might be varied and why?
ALU elements (lab # 22). Know what the three possible genotypes would look like on the gel. Why is only one ALU element (in the TPA gene) detected in this experiment? Be able to construct a simple pedigree based on ALU results.
PCR is a very useful technique in Molecular Biology. We used the PCR technique twice during the semester, once on our own DNA (to examine ALU) and once on the bacterial DNA samples (to amplify the rDNA fragment). In both experiments, the first step was lysing the cells (either bacterial or human). What is Chelex used for? Why is magnesium an important component in PCR? Why is it often added last? What were the similarities in the two protocols we used? Any differences?
Southern hybridization
(lab # 13)
Understand the principles behind
both Southern transfer and the non radioactive probe detection. Review
stringency and the conditions for low and high stringency. Review
nonspecific
binding and the use of a blocking agent. How does the non radioactive
detection
work?
Be able to locate which fragment
in a restriction digest should hybridize to the probe. How could we use
our data from the blots to determine which fragment from the gel was
detected? What can be possible reasons for multiple bands in a lane
when we expect only one?
Small oligonucleotides can be used
as probes (Southern, Northern, colony hybridization) or as primers
(PCR,
site directed mutagenesis). What are the important considerations for
binding
of an oligonucleotide to the template?
Review the presentation on "cycle sequencing". There is a link on our lab web site to this presentation. How is PCR used in the process? How does a capillary sequencer work? What are some things that will result in a poor sequence?
Understand the processes that we (and other researchers) use to identify organisms based on sequence data. Know how to read a chromatogram. For example:
What problems did we encounter
in our experiment? How could they be fixed or improved?
Finally, the lecture site has a link to
Dr Zimmerman's powerpoint under "links". This is a very comprehensive
summary of the poster project and would be good to look through as you
study this area. Please remember this is a HUGE file.
Scientific
Literature
We all read a sequencing
article, and we learned some interesting and useful things from the
article that helped in our experiment. Review the article
and the questions on the worksheet. Plus, groups found their own rRNA
article to present. We found many similarities between the author's
approach and the one we used for determining identity of unknown
bacteria.
Cloning
Be able to draw a cloning scheme
for cloning problems I propose, including (1) possible cloning
outcomes,
(2) if they would be detected in a transformed colony, (3) how you
would
detect them (antibiotic resistance? blue/white test?), (4) what the DNA
would
look like on a gel. (5) Be able to identify the main conformations
of uncut miniprep DNA (look over our lab gel photos and example
photos). Be able to draw a
gel and illustrate a positive clone and necessary controls for a
cloning
scheme that I propose.
Controls
We expect to
see proper controls
included in your answers.
Web
links to animations:
PCR: http://www.dnalc.org/ddnalc/resources/pcr.html
Southern Blotting: http://www.dnalc.org/vshockwave/southblot.dcr