Verification of Mutant Phenotype:

To verify that the mutant phenotype was cause by the plasmid in the cells, I shuttled the putative mutant plasmids from yeast to E. coli and then mini prepped the plasmids. I shuttled the plasmids from the four mutants that did not grow on galactose, the three that grew at a much slower rate than the controls, the mutant that was contaminated with mold, four mutants that grew only slightly slower than the controls, the control with the pBM258T/YTM1, and three controls containing the gap repaired pBM258T/YTM1 with each of the three unmutagenized inserts from the three PCR reactions. The transformation of the extracted yeast DNA to DH5-a cells was done with electroporation and yielded between 100 and over 1000 transformants for each sample. After mini prepping one transformant of each sample with the BioRad Kit, I transformed the plasmids back into yeast cells (JWY3400). I also set aside some of the mini prepped plasmids for sequencing.

The transformation to JWY3400 yielded more colonies than I could count for all but two of the transformations. One of these lower yield plasmids was a mutant that only grew slightly slower on galactose than the control and the other was a mutant that did not grow on galactose. These transformations still yielded over 50 transformants each. I patched three colonies from each of the transformation plates onto C-ura, grew the patches overnight, and replica plated:
1C-ura
2C-ura+2%gal
3C-ura+4%gal
4complete

After incubation at 30 deg C for two days, growth could be seen in all the patches, including those on the galactose media. In order to dilute out any glucose that may have still been present in the patches, I replica plated a second time:
1 C-ura
2C-ura+2%gal
3C-ura+2%gal
4complete

Again, I saw even growth in all the patches on all the plates except for the negative control (JWY3400). I decided to patch three more colonies from each transformation plate and replica plate again:
1C-ura
2C-ura+2%gal
3C-ura+2%gal
4C-ura+4%gal
5complete

I am still waiting to see if there is growth in these replica plated patches.