Lawrence P. Casson
lpcasson at phoenix.Princeton.EDU
Mon Jun 14 20:20:00 EST 1993
In article <1vfps5$afb at agate.berkeley.edu> mytelka at zenith.berkeley.edu ( Daniel Mytelka ) writes:
>Does anyone have a good protocol for Taq sequencing? I have been
>trying to sequence using the protocol in the PCR Protocols book
>(Innis, et al), but have been getting basically a huge smear down
>each lane with a few correct bands being a bit above background,
>and others being invisible.
>Anneal: 0.5 pm template and primer (I used 1, 2; my normal standards)
> 50 mM Tris 8.8, 7mM MgCl2
> Water to 10ul
> 70deg x 3'; 42deg x 10'
>Labeling: 5uCi dATP, 1.5 uM other dNTP
> 2U Taq
> water to 5.5ul (additional)
> 42deg x 5' (I did it at 37deg, because my primer melts around 45)
> Place at room temp (time unspecified; I did short)
>Termination: Add 4 ul to GATC tubes with 20uM each dNTP, 60/800/800/400
> uM of the ddNTP respectively
> Various temperatures x 5' (I did 37deg, 50deg, 68deg)
> Stop with 4ul stop solution.
> Heat 95deg, ice quench, load.
>The same primer and template work great in standard sequencing.
>Anyone have any clues or a different protocol?
> Dan Mytelka (mytelka at mendel.berkeley.edu)
It seems to me that this protocol is meant to be used following an
assymetric amplification reaction so that there are plenty of excess
copies present of the strand that you want your primer to anneal to.
I started experimenting with such a protocol and it worked great the
first time I tried it. Unfortunately, I have not reproduced such
results. I expect to try again soon. The time it worked, I got results
that looked far better than anything I ever produced with commercial
cycle sequencing kits. In particular there were many fewer ambiguities
where bands appear in more than one lane.
It seems to me that using end labelled primers is probably preferable to
including a labelling step as part of the sequencing reaction. There is
one less step to go wrong - especially if you are doing multiple sequences.
We have an automatic sequencing facility here that uses dye labelled
nucleotides. We run the reactions and they run the gels. Our lab has
obtained mediocre results at best from CsCl purified DNA. Since I have
gotten the asymetric amplification to work from bacterial colonies, I
will hopefully get around to coupling together these two protocols
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