Gap Repair:

I used gap repair to insert the mutagenized copies of ytm1 into the pBM258T/YTM1 plasmid. Because little is known about pBM258T, I had to test various restriction enzymes to find some that create a gap in the YTM1 insert. I tested the following enzymes that I knew cut in the YTM1 insert to see if they cut within the vector as well: SnaBI, AatII, SwaI, BsrGI, MluI, and XmnI. The only restriction enzyme that we have that cuts only in the insert and not the rest of the plasmid was SnaBI. PBM258T/YTM1 was digested with SnaBI to create linearized DNA (the enzyme only cuts once) and treated with alkaline phosphatase. This “gapped” plasmid was used in gap repair in yeast strain JWY3400 (ura-).

Three gap repair transformations were performed:

1. The first gap repair transformation used inserts created in the first PCR mutagenesis reaction. This transformation yielded zero transformants. Since there were a few transformants on the plate from the gap repair with the unmutagenized insert, I thought that the concentration of the mutagenized insert might have been too low for any results. I decided to repeat the transformation using 10ul of the various inserts instead of 5ul.

2. For the second gap repair transformation I used either 5ul or 10ul of each of the mutagenized inserts from both the first and second PCR mutagenesis reactions. Transformants were seen on most of the plates. There was no correlation between the volume of insert used in each transformation and the number of transformants. The number of transformants on each plate ranged from zero to approximately 300.

3. The third gap repair transformation used the inserts from the third PCR mutagenesis reaction that had been washed with PCI and EtOH precipitated. Transformants were seen on these plates as well. Again, the number of transformants on each plate ranged from zero to approximately 300. I did see the largest number of transformants resulting from the PCR reaction with the 20uM dCTP as compared to the other dNTPs.