MIDTERM EXAM Feb 20 - 22

This exam will cover both theory and practice of the techniques we have performed this semester. It will not include anything on 16s rDNA.

Review sessions:  Sunday Feb. 19 at 4 PM in the lab
                                  or Mon Feb. 20 at 6 PM in the lab
                                M,T,W sections have all covered the same topics.
                                We are both happy to answer questions from all sections!

Practical Lab Math answer key and additional lab math problems (with answers) will be on reserve in the library. A practice exam and answer key  will also be on reserve.  Note that this is an old exam and some experiments have changed.
 

Reading in Laboratory DNA Science:

All of "Basic Techniques", Labs 1, 2, and 3, pp. 3-53. Read discussion of antibiotic action, pp. 19-21.

Read pp. 76-77 of Lab 5; it contains important information on how cells become "competent" and what is thought to happen during the heat shock.

Read sections for Labs 6 to 8, pp. 89-140.

Read Lab 10, pp. 149-163.
 

Lab #1

Proper use of the micropipettors. Choosing correct micropipettor. Determining the volume setting. Proper care. How to draw up a sample. How to use the microfuge and other lab equipment properly.
 

Lab #2, Pre lab reading, safety handout

Use of bacteria in the Molecular Biology lab. Safe handling and sterile technique. How to streak plates (with a plastic loop!). Read the "Results and Discussion" section. It will help with the test, and also help interpret the transformation plates we obtained. Understand how antibiotics work. The Ampicillin and Kanamycin have different modes of action, and resistance is achieved in very different ways. What are "satellite colonies"? Also read "Results and Discussion" on Bacterial Culture Techniques (E. coli growth curve).
 

Lab #3 and Pre lab reading

Care and use of restriction enzymes. Math problems on diluting buffers or DNA. Know how to set up a restriction digest using stock DNA and reagents. Know what a Unit of enzyme activity is, and how much enzyme to use in a digest.

Review the principles of gel electrophoresis. How does ethidium bromide work (pg. 38)? What is the difference between tracking/loading dye and ethidium bromide?

Read through the protocol. Do you understand how they set up their restriction digests? Read Results and Discussion, and look over questions 1-9. We did Q#8  as homework. Make sure you understand that problem. A lot of people had trouble determining the fragment sizes from cleavage of Lambda DNA, especially the BamHI lane.
 

Lab #5

Read pp. 76-77, how cells become "competent" and what is thought to happen during the heat shock.
 

Lab #6 and Pre lab reading

Read Results and Discussion, understand what each step in the mini prep procedure accomplishes. This chapter has a much better discussion of the mini prep than Lab 10. Read discussion of DNAse and RNAse on pg. 100.
 

Lab #7

We are trying to make recombinant plasmid pAK, using cleaved pAMP and pKAN DNA. Be sure you do understand the principle and theory of this cloning and how ligation works. Remember that the restriction enzymes must be inactivated before the ligation is performed (why?).

Know how to determine the fragments that result from cleaving with specific restriction enzymes. Read pp. 111-112, 118-119, understand our cloning scheme and what bands to expect from pAMP and pKAN digestion. Know how to determine the possible combinations in a ligation. Read Results and Discussion, pp. 122-123, and be able to construct possible recombinants. Also know the difference between "sticky" and "blunt" ends. Understand that Ligase reforms phosphodiester bonds (Figure p. 121). Be able to draw out a "cloning scheme" like we did in class.

Lab #8 and Pre lab reading

After restriction digest and ligation, we have hopefully created some pAK recombinants. In exercise 8, we made bacteria competent to take up foreign DNA and then transformed our ligation mix (along with control DNA).

Read pp. 76-77, which explains what happens when bacteria become "competent". Read through the protocol and make sure you understand what we actually did, and why we did it. Review sterile technique and the mechanisms of ampicillin and kanamycin resistance. Know how to sterilely spread bacteria onto agar plates. What were the control plates we spread during this experiment? What would we expect to see on each of these plates? Why?

There is a lot of math associated with plasmid transformation (Results and Discussion questions 3-6). I did not have time to discuss them in class and I do not expect you to do these calculations.
 

Lab #10 and Pre lab reading (for Exercise 6 and Exercise 10)

Minipreparation of plasmid DNA. Chapter 6 has a good discussion of the two important topics:

• how the mini prep works and what each reagent accomplishes.
• the main conformations of uncut plasmid DNA

Uncut (mini-) versus cut (mini+) DNA:

Interpreting gels is an acquired skill in Molecular Biology. You should know what conformations to expect in uncut plasmid DNA; what RNA looks like on a gel (the "RNA cloud"); which fragments should be produced by digestion of pAMP or pKAN by HindIII and BamHI; what a partial digest might look like. Carefully read pp. 105-106. Study the gel photos on 107, 161, and 162. Also look at the previous gels and our gels from this semester. Interpreting pLIG DNA is the most challenging, but you should be able to identify which fragments originated from which starting plasmid.
Look at the control DNA on the mini prep gels, or look at the gels containing pAMP and pKAN digests we did for lab #7. These lanes all have the bulk purified DNA. Note the characteristics of mini prep DNA that we would not find in the bulk purified pAMP and pKAN.
 

Homework

Know how to determine the resulting fragments when DNA is cut several times with a restriction enzyme or enzymes:

• Lambda (linear and circular forms)
• plasmid (circular only)

Lab Math: know how to do the type of calculations you did in the homework. Remember you must show how you would actually make the solution; don't forget to bring solutions up to volume. Also, we add 1x  buffer (not water) to the agarose powder. Know how to make a buffer containing two or more stock solutions. Know how to set up a restriction digest.
 

Controls

Finally, as  discussed in class, it is important that all scientists use the proper controls in their experiments. For Molecular Biologists, this is critical because most of the time we cannot see what we are studying! So understand the use of controls in the procedures we have done so far this semester. Include the proper controls in your exam answers, or you will loose points.
Be able to suggest a proper positive or negative control. Be able to suggest possible errors in the experiment that can be detected by the controls (such as an inactive antibiotic resulting in growth on a negative control plate). Remember that "contamination" is a very vague and not acceptable answer. The Pre lab reading for Exercise 8  discusses controls for transformation and running gels, so this will be a good study guide.
 

Chances are very good that you will have a cloning problem of some type. However, it will not be pAK. Don't spend a lot of time memorizing that cloning exercise. Concentrate on the main ideas that you can apply to a new cloning problem.