difficult template

Bruce Roe BROE at aardvark.ucs.ou.edu
Mon Mar 26 08:59:34 EST 2001


Dear Sabi na Belli,
	Have you tried:
1. ABI's dGTP BigDye terminator mix
2. adding 5 or 10% DMSO to either the normal dITP containing BigDye terminator
   mixes or to the dGTP containing BigDye terminator mix.
3. attempting to PCR the region using the typical mixture of the 4dNTPs except
   replacing the dGTP with 7-deaza-dGTP and then using either of the above
   conditions to sequence the 7-deaza-dGTP containing PCR product.
4. 'spiking' the normal dITP containing BigDye terminator mix with the dGTP
   containing terminator mix (in a ratio of 4:1, 3:1, or 2:1 normal vs dGTPmix)
	We and others have found one of more of the above can be helpful in
reading through GC rich regions.
Cheers and wishing you good luck with this difficult region.
--B.Roe
*************************************************************************
Bruce A. Roe, Ph.D  George Lynn Cross Research Professor
                    Department of Chemistry and Biochemistry
                    University of Oklahoma, Norman, OK 73019-0370, U.S.A.
Phone: (405) 325-4912 or 7610;  FAX: (405) 325-7762;  e-mail: broe at ou.edu
********************** http://www.genome.ou.edu/ ************************

 You write: 
=> Date: Mon, 26 Mar 2001 08:56:34 +0100
=> From: Sabina Belli <Sabina.Belli at uts.edu.au>
=> Subject: difficult template
=> Sender: owner-autoseq at hgmp.mrc.ac.uk
=> To: autoseq at net.bio.net
=> 
=> 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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