Sequencing AT rich Templates

William Buikema buikema at ameritech.net
Tue Nov 11 23:52:50 EST 2003


We find that with bisulfite mutagenized templates, there are not enough
C residues left in many cases to hold the two strands together at the
standard extension temp of 60C.  We do these reactions with an annealing
temp of 54C, and see much better results.  You can also use the GTP kit
as an alternative, where the ITP in the standard kit is replaced with GTP.

Bill

Mick Jones wrote:
>
> Hi
>
> What is the best method to sequence AT-rich templates on a 3700 capillary
> sequencer.
>
> We have been given samples to sequence that contain ~800+ bp inserts in a
> standard pUC based plasmid.  The inserts have been generated by PCR
> amplification of bisulphite treated DNA (Cs gone to Us, 5meC remain as C) so
> they are very AT-rich.
>
> We seem to get good data on some templates, on others we get slippage.  Also
> the signal strength drops rapidly.
>
> Any advice gratefully received.
>
> Mick Jones
>
> Michael D Jones, BSc, PhD, ILTM
> Head Genomics Core Laboratory
> MRC Clinical Sciences Centre
> Hammersmith Hospital Campus
> London, UK
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