Yeasy PCR screening problem

Shoumo Bhattacharya sbhattac at mbcrr.harvard.edu
Thu Jul 13 10:49:46 EST 1995


Nigel Kenward <mbxnjk at vax.nott.ac.uk> wrote:
>Hi All,
>Any ideas on how to do PCR colony screening on yeast ? I've tried freeze 

>Very simple.  Using a yellow pipette tip, touch a yeast colony
and touch the tip to the side of your pcr eppendorf. You should
just see a faint white streak. Avoid the temptation of putting more
yeast. Add primers (1uM final), 10x Cetus buffer, 0.4 ul Taq per
100 ul reaction. Denature 95deg for 10min. Then 95x1', 40x1', 72x2'.
For subsequent 29 cycle use your primers optimized annealing temp. but
similar cycling. I found that one round at 40 improved results a lot.
Best of luck.
Shoumo




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