Sequencing Problem

Tom Knight via methods%40net.bio.net (by tk from shaggy.csail.mit.edu)
Tue Oct 16 21:18:20 EST 2007


"Solaiman, Dan" <Dan.Solaiman from ARS.USDA.GOV> writes:
> I would be most interested in learning more about this.  I had past
> experience of problem read-through when there is a hairpin structure.
> DMSO or betaine did not help me.  I had to manually read the weak
> sequencing peaks of the ABI output to barely make out the sequence.  If
> other people have better solution I would be grateful to learn more
> about it.  

You could also try raising the extension temperature of the sequencing
reaction from 60 to 66 or 68 degrees.  This would tend to destabilize
hairpins.  A cycle from 60 to 70 briefly and back to 60 again might
also work well.



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