short sequencing

John Ladasky ladasky at leland.Stanford.EDU
Tue Jun 10 00:53:05 EST 1997


In article <339CCF1D.53CF at jcu.edu.au>,
Lynn Hughes  <James, Cook, Universuty> 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.

	What sequencing chemistry are you using?  With Sequenase, you
can use the manganese buffer supplement supplied with the Sequenase kit.
This limits your reads to about 150 bp, and reads start quite close to
the primer -- I *think* you'll get your lower limit of 20 bp.

	I am not aware that any other sequencing chemistries can be 
altered for close primer reads.  You can play with dNTP:ddNTP ratios, I
guess.

	And what's the reason that you can't place a primer 30 bp farther
away from the sequence of interest than the one you are presently using?

-- 
Unique ID : Ladasky, John Joseph Jr.
Title     : BA Biochemistry, U.C. Berkeley, 1989  (Ph.D. perhaps 1998???)
Location  : Stanford University, Dept. of Structural Biology
Keywords  : immunology, music, running, Green



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