PCR sequencing

Mr Adrian Simon Turner rejuast at ucl.ac.uk
Thu May 19 03:44:44 EST 1994


Dear Netters
	I have been attempting some direct sequencing. The templates amplify
as single bands and are coding regions of known concensus. So far, all I
have obtained is a succession of blank films. I have been using one or other
of the PCR oligo's to prime, have tried both double and single stranded 
template (latter by asymmetric PCR or T7gene6 exonuclease) using a sequenase
& or a taq cycling seq.kit, trying both end labelled primers and 35SdATP 
incorporation. DNA isolation was by Promega Wizard from a gel (asymmetric)
or straight from the reaction, followed by a phe/chlor, etOH ppt, 2x 70%
etOH washes and drying at 37 C for 1 hour (I am aware of some netters'
worries about the use of guanidinium in the Wiz kit). Quantities were
verified by running aliquots on gels before subjecting the remainder to 
sequence analysis. Kit controls worked every time. Addition of aliquots 
of my DNA killed the kit controls, but given the lengths I have gone to
to to purify it I think this must be a DNA interaction. The only thing
I can think of that I haven't tried is a nested primer.
QUESTIONS
1. If the amp product is heterologous & comigrating am I right in thinking
the spreading of the bands the length of the lanes render it very 
2. Why should a nested primer be necessary? Surely if the amp produces
if you could mail them to me direct (adrian.turner at ucl.ac.uk). Thanks.
		



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