Sequencing Very AT Rich sequences on a ABI 3700 with BigDye

Mason, A.L. alm19 at leicester.ac.uk
Wed Oct 23 05:14:33 EST 2002


I concur with steve.

However, you might also want to consider using Amersham ET terminators; You need to calibrate the 3700 initially however.

To limit slippage, try cycling  ET terminators as follows:

95 30sec
55 4 minutes.............x30

This presupposes the Tm of your primer approximates to universal, i.e M13 conditions, so that combined annealing extension at 55C is appropriate;If not, introduce an intervening annealing cycle for your own custom primer using Tm - 3C for temperature but still append with extension cycle set at 55C for 4 minutes.

In the absence of BG 3.0 suggestion, hope this helps,


Regards,

Laurence S Hall
Cardiology Genome unit,
Leicester
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