question about PCR

Andrew Doherty A.Doherty at Bris.ac.uk
Mon Nov 11 03:41:16 EST 1996


Ahmet Arman wrote:
> 
> Hi netters,
> 
> I need suggestions from regarding my PCR.  I have  60 bp clones and I need
> single-stranded DNA from them.  I amplified my clones using my primers and
> ran them on the gel, I saw that there is 60 bp major band (expected size)
> and also big fragment. This means that primers amplified vector too.  My
> question is how can I get rid of big band.
> 
> Thank you in advance.
> 
> aa374885 at oak.cats.ohiou.edu
Hi Ahmet,

you could just cut your small band out of the gel and use it as a
template to run off ssDNA by cycling with only one primer. THe ssDNA
should then run just above your ds template and can be gel purified -
just a thought. I've done this to make very long PCR primers (up to
200nts!!!) for mutagenesis and it seems to work!

Hope it helps,
Andy D

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Dept. Anatomy			Tel (0117)9287421
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