FROZEN acrylamide sol'n!
joan at med.unc.edu
Thu Apr 18 12:01:34 EST 1996
tjackson at uoguelph.ca (Timothy R. Jackson) wrote:
>> we are making our own 8% denaturing acrylamide gel solution for
>> electrophoresis, sequencing, etc. and were told by a few people that it's
>> best to store the stuff in a 4 degree fridge. well our damn acrylamide
>> solution freezes!! some days it doesn't some batches don't - it's a real
>> pain! ... [rest deleted]
Duke Groebe <drg at prophet.pharm.pitt.edu> wrote:
>If you have urea in your gel stock, leave it out. *Everyone* knows that
>you're supposed to make urea solutions fresh anyway because the stuff
>breaks down. Make up a higher concentration of stock acrylamide solution
>(I think 40% is recommended) in H2O and dilute to make your final gel
>solution. So, for 50 ml of gel stock at 7M urea and 8% acrylamide, you'll
>need 21 g of urea, 5 ml of 10X TBE, 10 ml of 40% acrylamide solution (which
>is actually 38% acrylamide, 2% bis-acrylamide), and about 19.3 ml H2O. Mix
>under vacuum. Redi-2-use.
Actually, I make up our low-salt mix 2 L at a time and keep it in light-
tight bottles (covered with aluminum foil) at 4C for weeks at a time.
When we need to our a sequencing gel we pull a bottle out and get the urea
back into solution with a stir bar (always in the bottle) and plate. I've
poured and run hundreds of gels this way and have had very few problems.
It is important that the urea is all in solution but it generally doesn't
take all that long to get it in. I pull the bottle out before I put my
plates together and by the time I'm done it's in solution.
I think the original poster's main problem is not letting the urea go
completely back into solution before using it. Like I said, I've never
had a problem and I'm the one they come to when they want crystal clear
reactions and a pretty gel :).
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