difficult template

Bayliss, Stuart stuart.bayliss at csc.mrc.ac.uk
Wed Mar 28 03:12:10 EST 2001


We've had good results with the dGTP kit also, but start with DMSO for


Stuart Bayliss,
Genetics Core Facility,
MRC Clinical Science Centre,
Hammersmith Hospital,
Du Cane Road,
W12 0NN

Tel (lab): 020 8383 3181
Tel (office): 020 8383 8305
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-----Original Message-----
From: Y.F.LEUNG [mailto:yfleung at cuhk.edu.hk]
Sent: Tuesday, March 27, 2001 1:43 PM
To: autoseq at net.bio.net
Subject: Re: difficult template


You may want to try 1) adding DMSO in your sequencing reactions 2)
increasing the annealing temperature of sequencing reactions. If both don't
work you may consider trying ABI's dGTP bigdye kit which is targeted for
such difficult templates.

Good Luck,

Sabina Belli wrote:

> Hi!
> I am trying to sequence a 700 bp PCR fragment. I cannot seem to clone
> this fragment because it appears to be susceptible to rearrangements. I
> have managed to sequence about half of it, then the sequence just
> becomes illegible. Although I have used a number of forward and reverse
> primers, the sequence stops in the same place, at what appears to be a C
> rich region. I have been using the ABI system and dye terminators.
> Does anyone have any suggestion as to how I can sequence through the
> last 300-400 bp of this sequence?
> Thank you
> Sabi na Belli (PhD)
> Molecular Parasitology Unit
> University of Technology, Sydney
> Australia
> ---

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