Pfu

Robert Hartley rh at mblab.gla.ac.uk
Thu Oct 5 06:40:11 EST 2000


In article <8rhjj3$jau$1 at news.net.uni-c.dk>, "Ali El-Salanti"
<isl40117 at worldonline.dk> wrote:

Try this.

Keep you mix the same as the 500bp mix. The polymerase abviously works so
scale up the mix to 100ul and use this foas a cycle. 
> The PCR machine is a PE. GeneAmp 2400.
> The cycling goes as this:
> 
> 1. 94, 5 min
5min
> 2. 30 cycles of
>     94, 45 sec.
70 sec min
>     55, 45 sec.
60 sec @ 55   
>     72, 5 min
7 min @ 72
> 3. 72, 10 min
15 min @ 72

OK, this is overkil but give it a try until you get bands then decrease
time and increase temp to optimise.

Have you considered a Taq/deep vent mix for LD-PCR?

Cheers
Bob; Sunny Scotland
> 
> "Ali El-Salanti" <isl40117 at worldonline.dk> wrote in message
> news:8rhe4v$qp0$1 at news.net.uni-c.dk...
> > Hello.
> > I am having trouble with my Pfu polymerase. Template is genomic DNA and
> the
> > size of the product should be 2 and 3 kb. With the Taq polymerase I have
> no
> > problem amplifying the sequence, also when I try smaller bits (like 500
> bp)
> > I do get a product with Pfu. But I can not Pfu amplify the large sequnces.
> > Maybe it should be noted that the template i 80 % AT.
> >
> >
> >

-- 
Robert Hartley, Centre for Cell Engineering,Joseph Black Building
University of Glasgow, Glasgow G12 8QQ Tel: ++44 (0)141 330 4756, 
Fax: 0141 330 3730 mailto:rh at mblab.gla.ac.uk
Web : http://www.gla.ac.uk/Inter/CellEngineering






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