Dir. Seq. of small PCR frag.

Steve Steve
Tue Jul 22 08:11:40 EST 1997


In article <5ql8lf$fom at net.bio.net>, Charles Warren,
charlesw at u.washington.edu wrote:

> I am attempting to directly sequence a small (178bp) fragment on an 
> ABI377,
> and we seemed to get too much signal the last few reactions we have done. 
>  We
> are using whatever FS kit is not the absolute newest, but the one before 
> that.
>  We were thinking about either reducing the amount of DT mix, or reducing 
> the
> number of cycle sequecing cycles.  Anyone have any specific suggestions, 
> like
> how many cycles or how much mix?  Greatly appreciated.
> 
> TIA,
> 
> Chuck Warren, student
> Molecular Biomarker Lab, UW

Chuck, one thing I found when i worked running the core facility at
Caltech was small fragments will give very strong signals unless the
amount of template is reduced. The amounts of reagents and template in the
Standard ABD protocols were optimized for a 5 KB plasmid. You should
adjust the concentration down if tthe size of your fragment is
significantly smaller. The protocol reccomends 
0.2 ug/ul of the template DNA. If you go much higher than this you will
use up the reagents in the mix faster resulting in strong signal up front,
with shorter reads. Try cutting the concentration of your DNA by 1/10 and
try this and see what happens. Use the same cycling conditions and the
same amount of terminator mix. This worked quite successfully for me when
I was at Caltech.
Steve



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