Single Stranded DNA Sequencing

jrbrown at ac.dal.ca jrbrown at ac.dal.ca
Thu Sep 16 18:41:27 EST 1993


Re: double stranded vs single stranded sequencing.

I have had good success with both double stranded and
single stranded sequencing although the latter method
is more effect for eliminating compressions and for
sequencing high G+C templates.  The Taquence kit (from
USB) is the same price (or was the same price?) as 
Sequenase and utilizes the Taq polymerase.  Therefore,
higher temperatures can used for strand extension and
termination which, in turn, reduces secondary structure.

For single strand DNA preparation for sequencing, I use the
PEG precipitation protocal described in the Taquence and 
Sequenase manuals.  However, Taq polymerase is VERY 
sensitive to high salt and EDTA concentrations. Therefore
after precipitating the single strand templates in 95% EtOH,
I perform two 70% EtOH washes and then thoroughly vaccuum
or air dry the DNA.  Then, I rehydrate the DNA in sterile 
water, not TE.  I follow the sequencing method outlined in
the manual.  However, for both Sequenase and Taquence, I find
that limiting the extension times to 3.5 minutes rather than
5 minutes helps to reduce stops and compressions.

Hope that this posting is of some use and interest.

Jim Brown
Biochemistry Dept.
Dalhousie University
Halifax, N.S.
JRBROWN at ac.dal.ca   



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