Sequencing 2 year old PCR products.
Koen De Smet
k.desmet at nospam.ic.ac.uk
Thu Jun 25 02:14:23 EST 1998
Tim Beckenham wrote:
> Hi all,
> I need some advice on how to ensure good sequencing results using PCR products generated 2 years ago.
> The products are vector PCR amplificates [T7/SP6 primers (Promega) from the pGEM-T Vector system] in
> which 16S rDNA amplificates (27F/1492R) have been ligated. These were amplified from cloned cells, but
> alas, no stocks have been kept. These products have been stored at -20C for all this time, and I plan to use
> BigDye Terminating system for analysis.
> Any ideas would be greatly appreciated,as would direct email replies to my address.
> Thanks in advance,
> Tim Beckenham
Not exactly an answer to your question, but it may help you anyway: I
have done PCR on dilutions of amplified products that have been in a
-20C freezer for around 3 years. I have also done PCRs and restriction
enzyme digests on genomic DNA that has been frozen for similar periods.
I didn't have any problems that I would attribute to the DNA having been
stored for a long time and I think that very little can go wrong with
your DNA if it stays at -20C.
So I would just give it a go. Should the sequencing give problems, then
you can reamplify your PCR product and sequecne that directly.
Koen De Smet
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Imperial College School of Medicine at St Mary's
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