Sequencing query

Virginia Urquidi vurquidiI at molbiol.ox.ac.uk
Mon Jan 15 10:56:04 EST 1996


In Article <30F63332.4635 at nott.ac.uk>, Simon Dawson
<Simon.Dawson at nott.ac.uk> wrote:

>  I had an email from someone regarding the use of formaldehyde (formamide?) 
>sequencing gels to get round this problem. Has anybody else heard of this 
>method cuz I cant seem to get in contact with the person who suggested this 
>method via the email address they gave me. 

This method appears in Current Protocols in Mol. Biol.:

                               Acrylamide conc.(%)
                              
                              4          6       8
Reagent

Urea (g)                      42        42      42
38% acryl/2% bis (ml)         10        15      20
10X TBE (ml)                  10        10      10
Deionized formamide (ml)      40        40      40
H20 (ml)                      10        5       0

TOTAL VOLUME                  100       100     100

    (do not heat gel solution above 55oC)

Filter through Whatman No 1 paper

The gel sol. is viscous. Hold plates nearly vertical when pouring. Gel
should polimerize within 30 min.

Run the gel 45-70W constant power. This requires 60% higher voltage than
normal gels.  (in my experience, more than 2500V, so be sure you have a good
power supply) DNA migrates about half as fast.
Fix gel in 5% acetic acid/20% methanol (v/v).

Good luck.

Virginia Urquidi
NDCB
Oxford





               



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