Use of Salt Gradient in Sequencing Gels?

Pamela McLean pammie at acs.bu.edu
Thu Sep 16 12:37:40 EST 1993


In article <2756q2$2jd at charm.magnus.acs.ohio-state.edu>,
rrumpf at magnus.acs.ohio-state.edu (Robert Rumpf) wrote:

> I'd be interested in hearing protocols and experiences from those who have used
> salt gradients to improve sequencing gel results.  We have seen a protocol in 
> USB's editorial comments 19:2 page 64 that sounded intriguing, but wanted 
> second and third opinions.  Any help would be appreciated.
> 
> Bob


Hi,
We use salt gradients routinely in our lab. All you have to do is make two 
acrylamide gel mixes:

0.5X TBE Gel mix (1 litre):

150ml 40% acrylamide stock
50ml  10X TBE
460g  Urea


5.0X TBE Gel mix (1 litre):

150ml 40% acrylamide stock
500ml 10X TBE
460g  Urea
50mg  Bromophenol blue

Briefly, to 45ml of 0.5X mix add 72ul of TEMED and 25% ammonium
persulphate.
To 7ml of 5.0X mix add 11ul of each. Suck up 0.5X mix into 60cc syringe and
then carefully suck up the 5.0X mix being careful not to mix the two
solutions.
Pour the gel!!!

Hope this was of some use.

Pam

P.S. Takes approx. 3 hours at 1500volts (25 watts).



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