Design of dye terminator primers
myagi at u.washington.edu
Thu Nov 9 12:12:09 EST 1995
Our experience is that PCR primers work fairly well. The only thing
we've noticed is that, for some reason, primers with extra bases at the
5' end (e.g., restriction sites and a few extra bases) don't give as
clean results. They generally give readable signal for only 200-300
bases or so, rather than the 350-400 we see with other primers. Don't
really know why that is, just something we've seen.
On Thu, 9 Nov 1995, Bill Burnett wrote:
> (Dr. J.P. Clewley) writes:
> >Has anyone any hints or tips for the design of primers specifically
> >for cycle sequencing with an ABI dyedeoxy terminator kit?
> >Jon Clewley
> >Virus Reference Division
> >Central Public Health Laboratory
> >61 Colindale Avenue
> >London NW9 5HT
> >United Kingdom
> >jclewley at hgmp.mrc.ac.uk
> I just used my pcr primers initially, then a couple of internal 24-mers, I
> didn't do anything 'special', just less than 50% GC and a good 3' end
> preferably with a G or C at the end... I think ABI have an information sheet
> they'll send you but as I recall it doesn't say anything non-'standard'...
> If it's a terminator chemistry does the primer make any difference anyway?
> If someone knows otherwise could you post or email their words of wisdom?
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