Gradient Sequencing Gels

Bengt Oxelman bengt.oxelman at systbot.gu.se
Wed Jun 1 14:32:38 EST 1994


In article <2s2lkd$ina at mserv1.dl.ac.uk>, MACHADO <hide.machado at afrc.ac.uk>
wrote:

> 
> Dear Barry,
> 
> 	Salt-gradient gels can improve the number of bases that
> can be read. It's quite simple and I could read up to 400 bases
> in a good gel using a long and short run. Here the method:
>  	Make up the gel as usual (1X TBE). Start running the 
> gel with 0.5X TBE at the upper chamber and 1X TBE at the lower 
> chamber. When the blue dye almost reachs the bottom of the gel you
> can change the buffer (again 0.5X TBE top, 1X bottom) and load
> the samples for the short run. After 10 to 20 minutes add 1/2
> volume of 3M sodium acetate pH 5.2 (final concentration will be
> 1M Na.acetate). Run the gel until the blue dye (xylene cyanol)
> is about 8-10 cm from the bottom of the gel.
> 	
> 	If I'm not wrong the reference is:
> 
> SHEEN, J.-Y., SEED, B. 1988. BioTechniques 6(10), 943.
> 
> 	If you need more details e-mail me,
> 
>  						Hide
> 
> ==========================
> 
> H. B. Machado
> NFL - AFRC
> University of Sussex 
> Brighton   -   England
> 
> machado at afrc.ac.uk
> 
> ==========================

Yes, this works great! By loading the samples a second time and running c.
3h without sodium acetate and then 3h with 1M sodium acetate in the bottom
chamber we reach read lengths around 600 bases/reaction!
-- 
Bengt Oxelman
Dept. of Systematic Botany
Carl Skottsbergs Gata 22
S-413 19 Goeteborg
SWEDEN
bengt.oxelman at systbot.gu.se 



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