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Yeast Gene Disruption

Yeast Gene Disruption - Molecular Biology Techniques

Yeast Gene Disruption - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 05-11-2012, 12:35 AM
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Default Yeast Gene Disruption



Hi guys,
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!
Yeastnovice
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