ABI seq'g and DH10B cells

bckraev bckraev at aeolus.ethz.ch
Tue Jan 24 05:24:51 EST 1995


David, what I am going to say may sound a little irrelevant, since I have
never used myself the ABI sequencer, however, I do have considerable experience
with microsatellites... So, your clones in DH10B must be stable if they are in
plasmid vectors ( I checked the genotype ), but if they are made in M13, the
area containing microsatellite may undergo recombination. However, unique
sequences outside this region (e.g. polylinker) MUST be readable. I think,
your problem may rather come from cycle sequencing, since it may actually
produce artifacts, especially if your annealing step is below 60 C. If you are
using the standard 17-mer universal primer, you may try to switch to a 24-bp
primer (e.g. Promega) and cycle between 90 and 70 C. Hope this helps,

Sasha
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Alexander Kraev, Ph.D.                 Internet: bckraev at aeolus.ethz.ch
Lab. of Biochemistry III               Phone: 0041-1-632-31-47
Swiss Federal Institute of Technology  FAX:   0041-1-632-12-13
Universitaetstr.16
CH-8092 Zurich

"Some ideas are obscure not because they are complex, but because they 
 are excluded from our circle of comprehension" - Kozma Prutkov
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