5' overhang on PCR primers

Andrew Doherty A.Doherty at Bris.ac.uk
Wed Jan 29 03:52:22 EST 1997

Thomas F. Vogt wrote:
> I want to do PCR with a primer that has a 5'overhang of
> 50bp of unpaired nucleotides for the creation of an
> epitope tag. CAN this be done?  Is there a maximum number
> of nucleotides that can be left unpaired and PCR still
> work well.
THe answer to can it be done is - YES; one of the labs here regularly
puts the c-myc epitope tag onto various things using this kind of
protocol. I've recently done something similar withan even larger
overhang, and I still get a good PCR product. I'm now trying to
determine if it's the right stuff! However I found that the first few
rounds of the reaction had to be at a much lower annealing temperature
than that predicted for the whole primer. I started with 10 cycles at 42
deg C, followed by 20 cycles at 56 deg C. If you can get a product in
the early stages the you should be OK.

Hope it helps
Dr Andrew Doherty		email -  a.doherty at bris.ac.uk
Dept. Anatomy			Tel (0117)9287421
School of Medical Sciences	Fax (0117)9287402
University of Bristol
University Walk
Bristol UK

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