I am totally new in yeast work and I have a silly question to ask about gene disruption in Saccharomyces cerevisiae. I am trying to delete a gene in a haploid S. cerevisiae by using pUG-pSH (Cre-Lox) system. Here is what I tried so far:
1/ Construct PCR amplicons with 45 mer homologous sequences from upstream and downstream noncoding region of an ORF (obtained from Yeast Genome Deletion Website) using pUG plasmid as a template
2/ Gel purify the PCR amplicons (I am getting a non-specific amplicon about 500 bp even my annealing temperature is at 64 deg C)
3/ Transform into a competent cells with freshly made PEG3350
4/ Incubate at 30 deg C for 30 min and 42 deg C for 15 min (with DMSO between)
5/ Centrifuge to decant the supernatant and add extra carbon source to resuspend the cells
6/ plate out and incubate at 30 deg C
So I read a paper that it is important to have the same length of homologous regions instead of making their annealing temperature right. Is that true?
Thank you for your help in advance!