PCR product seq.
kraev at bc.biol.ethz.ch
Thu Nov 9 11:20:01 EST 1995
It looks like you put in too much template. In my experience, cycle
sequencing works in a rather narrow range of template concentrations,
and less template is very often better, then more ( unlike T7
polymerase). The kit protocols usually include a certain range, say,
5-100 fmol per reaction, which means that this often has to be tested,
particularly with PCR products. With a limited experience on the same
sequence, I can say that for a lambda template adequate picture is
obtained from 5 fmol, however, PCR products have to be tested from 25 to
100 fmol ( certainly not more ), with the same primer and cycling
conditions. It is assumed that a PCR product would reanneal more quickly
and that consequently each cycle is less efficient, but in fact it is
not known exactly why one sees this difference. Also, some PCR products
are capable of annealing on itself, such that they interfere with
progression of synthesis on the same strand from your labelled primer.
This produces sometimes strong bands in certain positions. However, the
latter event is rare, compared to stalling of polymerase at every tenth
base because of enzyme/nucleotide depletion with excess template.
Finally, insufficient polymerase activity is observed when cycling is
too slow, which happens with older cycler models quite often. Typically,
it should be 30 cycles made in 2.5 hours ( preferably less ).
I hope it does not discourage you from using cycle sequencing again.
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