mytelka at zenith.berkeley.edu
Sun Jun 13 12:58:29 EST 1993
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
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)
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