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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
Pipette Filler
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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!
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disruption , gene , yeast

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