PCR (and PCR Variations)

Biotechnology Chapter 4 Day 2 Study Questions

1. A thermocycler is an instrument that changes the temperature of your PCRs.  What is the purpose for each of the three cycled temperatures?
2. Why is the lowest temperature during the PCR cycling suggested to be in the 50-60C range “depending on the length and sequence of the primer?”
3. How would you determine the sequence of a cloned insert?
4. What is a wobble position and what does it have to do with degenerate primers?  When would you use degenerate primers?
5. How are the restriction sites generated at the ends of the template DNA when inverse PCR is to be used?
6. Draw the template and products for each step of RT-PCR.
7. Why would oligo d(T) primers result in a “3′ bias” of the original mRNA sequences?
8. How do you design PCR primers that add restriction enzyme sites to a PCR product?
9. Why can you clone the PCR product of some, but not all PCR reactions into a TA vector?
10. What do you do if your PCR product is not ready for TA cloning?  (This might happen if your PCR product (aka amplicon) is too long.)
11. What is the purpose of the three oligos in overlap PCR?  How does this differ with gene SOEing?
12. How can you control whether there is a deletion when an insert is made via recombination?
13. How can you design PCR primers that change a single base pair in a plasmid?
14. Draw the ribose sugar of rATP, dATP, and ddATP.
15. What did the original Sanger sequencing gels look like?  (This was back when radiation, rather than four colors, was used for sequencing.)