5' overhang on PCR primers

j.r.stephen mbi071 at sysc.abdn.ac.uk
Wed Jan 29 08:19:22 EST 1997


Hi Thomas,

For DGGE (denaturing gradient gel electrophoresis) you use a 5' "clamp" of about

60 G and C bases as standard. This doesn't interfere with the reaction and we
don't use any  
special conditions during amplification, my guess is that if something like that

does no harm, nothing will!


John
Andrew Doherty (A.Doherty at Bris.ac.uk) wrote:
: 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
: -- 
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: Dr Andrew Doherty		email -  a.doherty at bris.ac.uk
: Dept. Anatomy			Tel (0117)9287421
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