rh at mblab.gla.ac.uk
Thu Oct 5 04:03:53 EST 2000
In article <8rhe4v$qp0$1 at news.net.uni-c.dk>, "Ali El-Salanti"
<isl40117 at worldonline.dk> wrote:
> 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.
What Machine are you using and what are your cycle perameters.
Try increasing your extension time. I got this from the PCR manual that
ALKAMI.com proviode for free.
Pfu polymerase requires a minimum extension time of 1.5-2 mins/kb so your
extension time would be 6.5 mins on a slow ramping machine and about 5 on
a quick ramping (I'm allowing 30 sec for tube temp equilibration) however
you might even extentd these times and do it overnight.
Try this for a mix.
KCl 10mM (NH4)2SO4
Buffer @0mMTris-HCl (pH 8.2) or 10mM Tris-HCl pH8.8
Additives BSA,Triton x100.
I would recomend downloading this. I did and imply printed it and had it
bound for less than a pound.
Bob; Sunny Scotland. (yes, the rain HAS stopped) :-)
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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