Excess acrylamide in wells - (Apr/20/2009 )
I should had write "this may be a stupid question but..." but I'm a lazy person. 
And my question could be stupid because was the first thing I thought when I read the post and my first thoughts... well you saw what happen when I said them. 
Apologize the misunderstanding.
By the way it's her 
lol
BadKarma on Apr 23 2009, 11:33 PM said:
And my question could be stupid because was the first thing I thought when I read the post and my first thoughts... well you saw what happen when I said them.
Apologize the misunderstanding.
By the way it's her
lolNo dramas.
The exclamation mark threw me
Cheers
Hi
Try to wash the comb with Methanol before you insert into the plate , Add little extra TEMED and APS. wash the wells with water before you use them.
Hope it will solve and try to use the same spacers and comb of same size.
Regards
Sudhakar
Rob Steuart on Apr 20 2009, 08:57 PM said:
When i remove the comb from my stacking gel there is excess acrylamide in the wells blocking me from loading sample. The combs i am using fit snugly into the gel casette so not sure where the problem is arising. I never had any issues in teh larger system, has only started since i began using teh smaller system.
Any help would be great
Cheers
Rob
Rob Steuart on Apr 20 2009, 09:57 PM said:
When i remove the comb from my stacking gel there is excess acrylamide in the wells blocking me from loading sample. The combs i am using fit snugly into the gel casette so not sure where the problem is arising. I never had any issues in teh larger system, has only started since i began using teh smaller system.
Any help would be great
Cheers
Rob
Had the same problem as you couple of months ago, tried to wash the wells with dH2O but no good, the advise was all the same, wash the wells, i also placed the comb in Methanol. No use. Till i found a new recipe for the stacking Gel. Can send it to you if you still have problems
Cheers mate
Aris on May 8 2009, 07:29 AM said:
Rob Steuart on Apr 20 2009, 09:57 PM said:
When i remove the comb from my stacking gel there is excess acrylamide in the wells blocking me from loading sample. The combs i am using fit snugly into the gel casette so not sure where the problem is arising. I never had any issues in teh larger system, has only started since i began using teh smaller system.
Any help would be great
Cheers
Rob
Had the same problem as you couple of months ago, tried to wash the wells with dH2O but no good, the advise was all the same, wash the wells, i also placed the comb in Methanol. No use. Till i found a new recipe for the stacking Gel. Can send it to you if you still have problems
Cheers mate
Thanks Aris
recipe would be great
cheers
Rob Steuart on May 13 2009, 10:59 PM said:
Aris on May 8 2009, 07:29 AM said:
Rob Steuart on Apr 20 2009, 09:57 PM said:
When i remove the comb from my stacking gel there is excess acrylamide in the wells blocking me from loading sample. The combs i am using fit snugly into the gel casette so not sure where the problem is arising. I never had any issues in teh larger system, has only started since i began using teh smaller system.
Any help would be great
Cheers
Rob
Had the same problem as you couple of months ago, tried to wash the wells with dH2O but no good, the advise was all the same, wash the wells, i also placed the comb in Methanol. No use. Till i found a new recipe for the stacking Gel. Can send it to you if you still have problems
Cheers mate
Thanks Aris
recipe would be great
cheers
Here comes the recipe:
1.5 ml dH2O
625 ul 0.5 Tris
25 ul 10% SDS
400 ul 30% Acrylamide
12.5 ul 10% APS
5 ul TEMED
I used to always make the same % of bis-acrylamide stacking gel whatever was the resolving (sperating) gel's % with (some) successful results.
But then I read in Bio-Rad Mini-Protean's manual a a stacking gel recipe with variable % and figured that it should match the resolving gel's %.
I asked around: some confirmed this assumption while others said that an intermediate % (e.g. 10%) was fine with any resolving gel.
Anyway, now i follow Bio-Rad's instructions just to be safe...
and even if it turns out that washing the wells was not your (main) problem, I assume it's always best to do so with ddH2O.
My thinking is you don't want the extra liquid (whether it be acrylamide or not) after polymerizing to mix with samples.
Tfal on Jul 8 2009, 11:43 AM said:
My thinking is you don't want the extra liquid (whether it be acrylamide or not) after polymerizing to mix with samples.
it's best to use electrode buffer to wash the wells.
Hey mdfenko, thanks for the advice. I guess pH wise it better to use electrode buffer...
Is it good enough if I just rinse the wells briefly with ddH2O and then fill them with electrode buffer when I set my cassette (gels) in the electrophoresis container?
