HELP! PCR using pfu
andybean at cmgm.stanford.edu
Wed May 10 13:40:03 EST 1995
In article <johnaris-1005951329590001 at 126.96.36.199>,
johnaris at anatomy.med.ufl.edu (John P. Aris, Ph.D.) wrote:
> Dear colleagues,
> I have been using pfu DNA polymerase to amplify a 3-kb fragment from yeast
> genomic DNA. I tried different conditions:
> 94 oC 1.5 min
> 45,50,55,60 oC 2 min
> 72 oC 6,7,8 min
> 10 and 100ng template
> 2.5 and 5 units
> for a total of 50ul reaction. Each time I used taq polymerase as a control.
> I could obtain a 3-kb product from taq but nothing from pfu. If anyone knows
> how pfu works, would you please tell me about it? Thanks.
> Pei Wu
> Anatomy and Cell Biology
> University of Florida
We have amplified a 4 kb product by using a mix of Taq:Pfu in a 3:1
ratio. The idea is that Taq does most of the work but you have Pfu in
there for proof-reading.
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