difficult template

Y.F.LEUNG yfleung at cuhk.edu.hk
Tue Mar 27 07:42:33 EST 2001


Hi,

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,
Y.F.

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
>
> ---

--
Y.F.LEUNG
Department of Ophthalmology and Visual Sciences
Chinese University of Hong Kong
3/F Hong Kong Eye Hospital
147K Argyle Street, Hong Kong
Tel: (852) 27623152 Fax: (852) 27159490
email: leungyukfai at hotmail.com, yfleung at cuhk.edu.hk
URL: http://i.am/yfleung (My functional genomics home)





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