Pcr sequencing

Mike Poidinger skspoidn at susssys1.rdg.ac.uk
Wed Apr 8 10:10:05 EST 1992


(coming late to the conversation, in response to <9204071426.AA04494 7/4/92>)
I have used, and got clean sequence results with, the Promega
PCR sequencing kit, which utilizes modified Taq polymerase. I have used
5' end labelling with 32P of PCR primers and internal sequencing primers.
PCR product (800bp from genomic DNA) was milk-glass cleaned, and small
 quantities (50 fmol) were
sequenced according to product specifications. Sequence is (sort of :-) readable
 to
within a few bases of the primer.

I have found that the cycling protocol depends on the thermal cycler being used,
especially wrt whether it measures plate or tube temperatures. Setting the
temp control to tube can mean long wait periods at plateau temperatures before
the cycle countdown begins.

 (I THINK:) If a low (<55 deg) annealing temp is used, Considerable
strand elongation and termination can occur during this temp stabilization
 period,
and at this temp, the system is sensitive to secondary structure false
 termination,
resulting in strong cross banding, poor sequence etc. Taq can incorporate 90
 bases/min
at 37 deg

Idealy, annealing temp should be a few degrees below melting temp, and should
 only
be held for 1 minute at the most. I use 30 secs.

Promega kit, including ddNTP's, PNK, Taq pol for 100 reactions costs 117 pounds
 stirling



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