Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum

Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum (http://www.molecularstation.com/forum/)
-   Molecular Biology Techniques (http://www.molecularstation.com/forum/molecular-biology-techniques/)
-   -   Yeast Gene Disruption (http://www.molecularstation.com/forum/molecular-biology-techniques/85336-yeast-gene-disruption.html)

yeastnovice 05-11-2012 12:35 AM

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


All times are GMT. The time now is 12:36 AM.

Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved

Page generated in 0.08830 seconds with 11 queries