PCR product seq.

[Karl Voss]

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


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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