Problem using pfuturbo for PCR

moez torki torki at NOCS.TSUKUBA-NOC.AFFRC.GO.JP
Wed Jun 16 22:05:06 EST 1999


Hi Alan,
I don't really have an explication for your troubles, but just a bad
experience with Stratagene's Pfu (I don't have experience with TurboPfu).
When I used Stratagene's Pfu I could observe smaller contaminants bands
when the expected products exceeded 2.5 Kb, and could not amplify enough
product when it exceeded 3 Kb. I switched to Promega's Pfu and I am really
very happy with the results : Great amplification (even up to 8 Kb product
from plasmids, phages and genomic DNA), high specifity and high fidelity.
That polymerase works so fine, compared to Stratagene's, that I wondered if
it was really proofreading one... And I am not working at Promega Corp :-)
BTW did you try to ampilfy with only one primer at a time ? maybe one of
your primer is sticking to an inverted reapeat somewhere on your plasmid.
Good luck,
Moez

PS. I use 300 mM dNTP, 0.5 microM primers, 2 units/100 micoL and 3 min/Kb
extension.

_________________________________________
Moez Torki, Ph.D.
Plant Biotechnology Institute,
Ibaraki Agricultural Center, 3165-1 Ago,
Iwama, Nishi-Ibaraki 319-0292, Japan

Tel:   +81-299-45-8330
Fax:   +81-299-45-8351
Email: torki at nocs.tsukuba-noc.affrc.go.jp
_________________________________________
The World : once thought to be flat, then proved to be round. Now it's
quite definitely Web shaped....





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