AUTOMATED PCR SEQUENCING

mwatson at uniwa.uwa.edu.au mwatson at uniwa.uwa.edu.au
Fri Nov 18 13:27:17 EST 1994


Dear Netters

This is aimed at those of you who may be familiar with the ABI 
(Perkin Elmer) Automated sequencer.  I am having trouble getting PCR 
products to sequence.  I PCR them to a clean single band and then 
NH4Ac precipitate.  I get sequence which cuts off at the correct 
size but it is just very muddled and makes very little sense.  My 
control sequence works fine.  I have tried increacing annealing in 
the sequencing reaction to no advantage.  I was wondering if anyone 
out there had any views and alternative purification methods for the 
PCR products which are 280bp long.  I do not think there are any 
primers left over from the PCR since sequencing with no primer added 
gives no signal at all.

Please reply direct

Cheers all
Mark Watson



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