PCR product seq.

[Rover]

Victor.A.Kim at dartmouth.edu (Victor A. Kim) wrote:

>Hi!

>I was wondering if anyone out there could help me with a sequencing
>problem I have been constantly experiencing.  I have been trying to
>directly sequence a 200 bp PCR product using New England Biolabs
>thermal cycle sequencing kit.  I can read patches of the sequence
>clearly, but every so often I get bands across all 4 [GATC] lanes
>making those regions of the sequence unreadable.  I have already
>subcloned and got a clear sequence of this PCR product, but I would
>like to *directly* sequence the PCR product so I can avoid subcloning. 
>The PCR product is pretty clean in my opinion [gel purified] so I don't
>think it's the quality of the PCR template.  I have also tried using
>nucleotide analogs such as deaza G and deaza T and all I get with those
>are huge smears on the gel.  I'm thinking now that perhaps cycle
>sequencing is too sensitive for PCR product sequencing?  Thanks for
>your input!

	We also sequence PCR products, and have had similar problems.
We can usually alleviate it by using longer primers with a higher GC
content and by raising the annealing temperature, or using none at
all. Cycles of 95 (30 sec) and 70(1 min). Everything I have read
though demonstrates that cycle sequencing small PCR products, such as
yours, is poor at best. Grin, I have also generated my own truely poor
sequences.

	I would be interested if anyone  has had great success sequenceing
small PCR fragments
Rover at livnet.com
Barlow at picard.evms.edu (The Mad Molecular Biologist)