PCR sequencing

Paulo Magalhaes pamaga at biobase.aau.dk
Thu Jul 16 07:26:30 EST 1992


HELLO OUT THERE !!!

Did someone read my request for help on PCR sequencing ?!

I said I was trying to set up a protocol for "dsDNA cycle sequencing",
using a double stranded PCR product as template, a 20mer unlabeled oligo
as primer, a mixture of the 4 dNTPs, 35S-dATP and, finally, ddNTPs.

IT WORKS !!! My only problem is that my A and C bands are faint,
especially for the higher molecular weight fragments. Although I am
grateful for the only reply which I saw posted , I don't want a kit:
what's so difficult about making a mixture of the 4 dNTPs ?!?! - they
already come in solution, it's only a question of diluting them...
The same applies to the ddNTPs.

Going back to the problem, then, I think the reason for my faint bands
is a "bad" d:dd ratio - the growing chains are terminating too soon.
If this is the case, I should be able to solve the problem by trial and 
error (ANY OTHER OPPINIONS about the reason for the faint bands are
welcome, by the way); what I was trying to do when posting my request 
for help was saving some time and a few gels.

I got a direct reply in my mail box suggesting the use of 33P (I had
said that I didn't want to use 32P), but this is avoiding the issue:
I am grateful for the suggestion, but what I want to find out are the
optimum d:dd ratios for the 4 nucleotides.

I would also appreciate any data on thermal stability of ddNTPs. Is it
because they get degraded that one has to use such high concs ? Or is it
simply because the traditional "labeling reaction" is running in parallel
with the "termination reaction"?

If anyone has ANY information that will contribute to my understanding
of this problem, please share it. Hint: this time my fax no. is at the 
end of this note, Mike! More then having an aesthetically pleasant gel, 
I would like to KNOW why it turns out so beautiful...

Thanks again for all your help,

Paulo

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Paulo Magalhaes        |         Tel: +45 / 35 45 45 92
Rigshospitalet - 4062  |         Fax: +45 / 31 39 65 43
Blegdamsvej 9          |
DK-2200 Copenhagen     |
Denmark                |  e-mail: pamaga at biobase.aau.dk
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