general questions about sequence with BIG-DYE kit (ABI-system)

[P.J. v. Santbrink]

I want to sequence a insert in a vector with the Big-Dye kit from Perkin
Elmer.(I'am rather new in the sequence world)
I've got some general questions:

-    the cycle conditions state no initial denaturation (2-5 MIN. 95oC)
step. why not ? I ask this because when I perform a "normal" PCR
reaction and I leave out this initial step the intensity of the band is
lower. So can I use this step in the sequence reaction without
disturbing the reaction somehow.

-    also, the annealing time is only 5 seconds: we've got a rather old
PCR-apparatus, should I lenghten the time (same for denaturation time of
10 seconds).

-    I want to perform the cycle reaction overnight , I read that people
then use  99 cycles. should I do this

-    which conditions should I vary (concentration DNA, primer
concentration, amount of reaction mix)

-    the protocol states to use 8 ul of the mix. I've read that people
use much lower quantaties. this cuts in the costs but what will this do
to the signal intensity


There are probably more things to think about (outside the ones
mentioned in the ABI protocol) but this is a start

Hope some sequence goeroes can help me

Peter van Santbrink-

-
Division of Biopharmaceutics
university of  Leiden
The Netherlands
E-mail: santbrin at LACDR.Leidenuniv.nl