Cycle sequencing of PCR products

hpotter at chmeds.ac.nz hpotter at chmeds.ac.nz
Sun Aug 7 16:45:06 EST 1994


In article <kwan-0508941200180001 at 132.206.101.240>, kwan at medcor.mcgill.ca (Tony Kwan) writes:
> Hello Netters,
> 
> I am in the process of sequencing a large number of PCR products all
> having some kind of point mutations or other and we've decided that the
> best way would be to amplify the piece directly from a yeast colony,
> purify the product and use cycle sequencing to obtain the sequence.  We've
> just tried the New England Biolabs CircumVent kit and the sequence is not
> as clear as we would hope for.  Has anyone out there has any success
> sequencing PCR products directly using any of the other kits available
> commercially?  Any feedback would be helpful.  Thanks in advance.
> 
> -- 
> Tony Kwan
> Department of Biochemistry, McGill University
> kwan at medcor.mcgill.ca

Tony,
I have found the Life Technologies,Inc dsDNA Cycle Sequencing System to be an 
excellent kit for cycle sequencing PCR products. I use gamma 33P ATP from
Amersham for end-labelling primers. The secret to nice easy-to-read sequence
with minimum background is to keep both the amount of template and the numbers
of cycles to an absolute minimum; typically, for a 200-300bp product, I use
20 fmoles of PCR product and perform 20 cycles using 20 seconds denaturation,
20 seconds annealing, and 20 seconds extension per cycle. Sequencing reactions
are run on a wedge gel. I find that with a double loading I can get 200 to 250
bp of readable sequence no problem! With a really good run you should be able 
to read sequence within 10 bases of the primer which means you can use the 
amplification primers for sequencing. Hope this helps and good luck.

Howard Potter
Molecular Pathology Group
School of Medicine
Christchurch
NEW ZEALAND     



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