PCR Mutagenesis:

PCR mutagenesis was used to create single point mutations in the YTM1 gene. I first tested my primers by doing a PCR reaction with the normal reaction conditions (200uM dNTPs, 600nM primers, 1ul of a 1:10 dilution of mini prepped template plasmid DNA, 1x buffer, and 1ul Taq enzyme). Analysis on an agarose gel showed that the PCR worked fine.

1. The first PCR mutagenesis reactions contained 10-fold molar less (20uM vs. 200uM) of one of the four dNTPs. Each of the four possible reactions was run along with a control containing equal molar concentrations of all four dNTPs (200uM). The other reactions conditions were standard. I repeated my PCR cycle 18 times.

2. The second PCR mutagenesis reaction contained the same reactions conditions and the same PCR program. This time I repeated the PCR cycle 20 times in hopes of getting a larger yield of mutagenized insert. However, a larger number of PCR cycles increases the possibility of having multiple mutations within each copied insert.

3. The third PCR mutagenesis contained template DNA (the plasmid pBM258T/YTM1) that had been washed with PCI and EtOH precipitated. The rest of the reaction conditions were the same as the conditions for the first two PCR mutagenesis reactions. I also used a longer elongation time—3 minutes vs. only 1 minute for the first two PCR reactions. Analysis on an agarose gel showed that more product was present with the longer elongation time, especially for the reaction containing 20uM dCTP.