PfuTurbo (hotstart version)
fw218 at cam.ac.uk
Mon Nov 12 12:49:52 EST 2001
Has anyone troubleshooted this enzyme, successfully or not? I am using
60mers to amplify 2-3 kb. 80% of my reactions yield zero product on a
gradient cycler. All of them work beautifully with Taq. I follow the
manual in terms of dNTP and primer concentrations, and I use an
extension time of 1 min/kb. My primers are simply desalted, not (HPLC)
purified. Stratagene rep claims this could make a difference but I don't
see how since purification only removes truncated oligos and the 5' tail
of my primers isn't complementary to the initial template anyway.
Stratagene rep is otherwise incompetent. Control reaction amplifying 1
kb also with 60mers works fine.
Any suggestions? I've already burned through 250U so I'm somewhat
reluctant to keep changing parameters without effecting any improvement.
If public opinion is that this enzyme sucks for long amplicons, what
alternatives are there? I have previously used Pwo (Roche) but have been
unable to find out much about its error rate compared to Pfu. I am not
going to be able to sequence, so I figure Taq/3'-5' exo mixes aren't
Thanks in advance!
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