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PCR product seq.

Karl Voss karl at hobbes.chem.ualberta.ca
Wed Nov 22 15:27:17 EST 1995

Sasha Kraev (kraev at bc.biol.ethz.ch) wrote:

> Victor,
> 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.

We have certainly noticed that the concentration of template DNA is crucial.
We get pretty good sequences with Thermo Sequenase kit and aproximatly 20ng
of a 700bp template PCR per color (ABI fluorescent primer set).  We measure
our template concentrations by ethidium bromide fluorescence in a filter
flourimeter.  I would certainly recommend that you do this if at all possible.

Good luck


Karl Voss
Department of Chemistry
University of Alberta
Edmonton, Alberta, Canada
T6G 2G2

karl at hobbes.chem.ualberta.ca
phone 403-492-0222
fax   403-492-8231

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