PCR amplification with one long primer

N. Linford nantzee at u.washington.edu
Tue Jun 24 17:29:40 EST 1997


I have been running into some trouble getting a particular PCR reaction to
work. I am using the product of a previous amplification as one of my
primers (250 bp) and then a 60mer to prime in the other direction. I have
been using PFU polymerase, 100uM DNTPS, 50ng template, and varying ratios
of each primer. I am trying to amplify a 700 bp region. I have tried a
standard PCR reaction (95 1min,60 1min,72 1min) and a two step PCR
(excluding the low temperature anneal). I cannot seem to get any
amplification to occur. I have checked the DNTPs, enzyme, etc and they 
work in other types of reactions. Does anyone have any ideas on how
to convince my long primer to anneal to the template and amplify my
piece?
 
Any help would be appreciated
(please email me a copy of replies)
nancy
_______________________________________________________________________________
Nancy Linford					UW Dept of Pharmacology, 357280
nantzee at u.washington.edu			f-428       543-8502
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