sequencing from 300-500 bp

Michael Holloway ca566 at cleveland.Freenet.Edu
Mon Nov 23 13:13:14 EST 1992


In a previous article, mm6y+ at andrew.cmu.edu ("Morris F. Manolson") says:

>Dear Netters,  I am sure someone must have asked this before but......
>
>I am trying to sequence off of one reaction 300 to 500 bps using the 
>standard IBI or BRL 44 cm glates.  Without having to resort to ultra-long
>plates (of which I don't have), does anyone have any suggesting on how to
>improve the resolution of the bands in the 300 to 500 bp region.  My reactions
>are fine and I can see bands up to 600 bp from the primer, I just can't
>resolve them well enough to read.  I am using 6% acrylamide wedge gels and run
>them for 24 hours at 900 Volts, this gives me nice sequence on average
>up to 350 bp,  on a great gel I can get 400.  I remember some company trying
>to sell an alternative matrix to acrylamide, (of course I don't remember
>who or where) which they claimed improved resolution, does anyone
>out there know what I am talking about?  Thanks in advance, Morrie 
>

Based on my experience (and I think the other replies to your questions
bear me out in this) the best advice I can give you is not to try.  Bite
the bullet, make or contract out for an oligo and prime from within your
last bit of sequence.  Even if you do manage to get that last little bit of
sequence out of it the quality will be extremely low.  I've been there and
I can honestly say that I wish I hadn't gone.

Mike
-- 
E-mail: mhollowa at ccmail.sunysb.edu (mail to freenet is forwarded)
phone:  (516)444-3612



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