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Bio312 Week 3 Lab Reports: Key and Comments

(The correct answers to questions 1 and 2 varied by group.)

3. Why did I incoporate two different annealing temperatures in the PCR protocol?

(student answer) The incoporation of a lower annealing temp during the first 5 PCR cycles was necessary because in the beginning cycles of PCR we are dealing with primers carrying uncomplementary tails at their 5' ends. Before these tails are incoporated into the DNA (in later PCR cycles) the number of bases pairing up between primer and template are fewer and thus have lower Tm values than in later PCR cycles. The annealing temp (5 degrees below Tm) would therefore need to be lower in the earlier cycles. It's important to have as low an annealing temp as possible so that primer and template will have the best possible chance of annealing, but not so low that the primer loses its specificity for its complementary template sequence.

4. Determine both relevant Tm values for each of your primers, using the formula

Tm = 4 (# of GC basepairs) + 2 (# of AT basepairs).

Both means the Tm for the primer hybridizing with the original template and the Tm for hybridization in later rounds, when PCR products are serving as templates. In the first case, only the bases of the primer that can basepair with the genome should be considered. In the second case, the entire primer sequence applies.

Several groups noticed that this "rule of thumb" formula is not terribly accurate with very long primers such as the ones we're using. The estimates can be ridiculously high (e.g. over 100 degrees C). That's why I used the more complex formula available on the web for doing the estimations for lab.

5. Why do you think the rule of thumb is that annealing temperatures should be 5 degrees celsius cooler than the lowest relevant Tm?

(student answer) The Tm is the temperature when 50% of the primers and DNA fragments have annealed together. Subtracting 5 degrees allows for more than 50% to anneal together. We don't subtract more than 5 degrees, because lower temperatures reduce the accuracy required in annealing. The inaccuracy comes from primer dimer formation, primers annealing to spots on the template DNA that are not completely complementary, and DNA reannealing to itself.

(The answers to 6 and 7 varied by group.)

8. In two weeks, you'll be cloning your PCR product into a pCITE vector. To do this, you'll trim the PCR product at the ends, using restriction enzymes that will allow cloning into pCITE. Here are the recognition sites of a few of these enzymes. Which of them could you use?

All of the "direct" primer tails can be cleaved with NcoI, and all of the "reverse" tails can be cleaved with either AvaI or XhoI.

Several of you expressed concern about cutting the "direct" end of your product (i.e. the 5' end of your gene) with NcoI, because its recognition site includes the start codon ATG. However, you will also be cutting your pCITE vector with NcoI. This will give the following structure:

pCITE vector:

C
GGTAC

PCR insert (the underscores are meant to represent blank spaces):

CATGG...
____C...

When these two ends anneal together and are then ligated to each other, the original sequence of the recognition site is restored, including the ATG. (This is a general feature of recognition sites, as long as both of the ends were generated by the same enzyme.)

Those of you cloning either LTV1 or YOR021C also pointed out that the creation of this NcoI site involved introducing a mutation into the second codon of your gene. In LTV1, it was changed from TCG (Ser) to GCG (Ala). In YOR021C, it was changed from AAG (Lys) to GAG (Glu). We will need to keep these changes in mind as we continue our project. Our hope is that they have no impact on the structure and function of the gene products. This is true for most single amino acid substitutions, although not for all. Small changes to the sequence are sometimes unavoidable, however.

9. For this "trimming" to work, what needs to be true of the frequency with which your enzymes cut within the gene sequence you're trying to clone?

This frequency should be zero.

NOTE: you need not use probability to determine this, since the sequence is known. Just scan the gene's sequence for the recognition site you're interested in. (I've already done this, using a software program designed for the purpose. You'll be using this same software later in the semester.)

10. For the RPS3 and YAR1 constructs, there were two reactions in addition to the "no template" control. One used a plasmid template and one used a genomic template.

a. What's the main advantage of using a plasmid template instead of a genomic template?

(student answer) The plasmid template is preferable simply because of its smaller size. The chance of the primers amplifying other parts of the template that have some sequence homology is very low with the plasmid. This may not be true of the genomic template.

b. Why do we need to do these PCR reactions at all, given that we already have plasmids that carry these genes?

(student answer) Even though we already possess a plasmid with our gene inside, we want to perform the PCR reaction and insert the gene into a different plasmid to enable us to attach a tag on our protein. This will allow us to separate the protein from extraneous biological matter within the cell. The importance of using PCR prior to inserting the gene of interest into a different plasmid is the ability to manipulate the ends of the gene. In our case, we are adding restriction sites to the ends of the gene, which allows us to splice the entire gene into the plasmid. By inserting the gene into a vector with a T7 promoter at the beginning of our gene, we are capable of getting high expression.

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