In article <4ajlbc$plq at oracle.rz.uni-ulm.de>, wagner at lyra.rz.uni-ulm.de
(Franz Wagner) wrote:
> I am trying to sequence several alleles consisting of very AT-rich sequences.
>> I am using an ABI 373 sequencer. The alleles are subcloned into pMos.
> I used both T7 dye terminator and cycle sequencing dye terminator sequencing,
> but I do not get usable sequences. T7 sequencing failed totally. With cycle
> sequencing, A and T peaks are getting smaller soon, C and G peaks are very
> high, and the total reading length is about 200.
Try using ABI's AmpliTAQ FS Dye terminator Cycles sequencing Kit. The new
enzymes give results similar to Sequenase, without have to start with a
huge amount of template. I have done this in my core facility at CalTech.
The new enzyme is more efficient at incorporating the Dye labeled
ddTerminator. You don't even need to use spin columns for the clean up.
NaAcetate in ETOH precipitation is sufficient. You will get between
500-600 bp reads from a clean template prep. Keep the template
concentration close to 0.2 ug/ul for the best results. Hope this helps.
California Institute of Technology DNA Sequencing Core Facility
email: marshs at cco.caltech.edu (this post came from my home account)