short sequencing

Hiranya Roychowdhury hroychow at NMSU.EDU
Wed Jun 11 00:18:59 EST 1997


At 01:50 PM 6/10/97 +1000, Lynn Hughes wrote:
>Hi all,
>
>Just hoping for some suggestions with a sequencing problem we are 
>having.  We are wanting to obtain the sequence of our DNA about 20bp 
>from where the primer attaches.The closest we have achieved so far is 
>about 40bp.  Has anyone got any suggestions on how to get closer to 
>the primer attachment region.
>
>Thanks for your help.
>
>Lynn Hughes
>Dept of Biochemistry and Molecular Science
>James Cook University of North Queensland
>Australia
>
>email  lynn.hughes at jcu.edu.au
>
>

These are the steps that may be useful:

1. Use double the amount of template that you normally use.
2. Use double or 1.5x primer than you normally use.
3. Do the labeling and termination reactions for 2 min each.
4. make sure that the labeling reaction is carried out slightly below room temp.( 20 C)

With these manipulations I have been able to read within 10 bases of the primers. 

Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu



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