gel electrophoresis - problem with stacking (Jan/18/2007 )
we store eliquots at 4C and they always work.
I thought APS have to be made fresh. Thought it will be degrade after some time.
hi all,
i also have had another problem with the stacking gel today;
I cast two gels at the same time (from the same stock mixture) treated them exactly the same, cleaned the glass plates the same etc etc. One gel set just right with no shrinking seen at all, but on the other gel, the wells had shrunk so badly that it was unusable!
I often have shrinking problems, and have tried fresh APS and increasing the volume, and fresh temed etc. It just seems a random process to why it shrinks sometimes and not others.
Does anyone have any ideas about this? People always just seem to say fresh APS and fresh temed, but this doesnt help!
Thanks for any help you may have, its driving me mad!!
(stack = 7.35ml water, 1.25ml 6.8 1M tris-hcl, 1.3ml 30% bis-acrylamide, 100ul SDS, 50ul APS (or up to 100ul no difference) 10ul temed)
i also have had another problem with the stacking gel today;
I cast two gels at the same time (from the same stock mixture) treated them exactly the same, cleaned the glass plates the same etc etc. One gel set just right with no shrinking seen at all, but on the other gel, the wells had shrunk so badly that it was unusable!
I often have shrinking problems, and have tried fresh APS and increasing the volume, and fresh temed etc. It just seems a random process to why it shrinks sometimes and not others.
Does anyone have any ideas about this? People always just seem to say fresh APS and fresh temed, but this doesnt help!
Thanks for any help you may have, its driving me mad!!
(stack = 7.35ml water, 1.25ml 6.8 1M tris-hcl, 1.3ml 30% bis-acrylamide, 100ul SDS, 50ul APS (or up to 100ul no difference) 10ul temed)
perhaps the answer is in the combs... I don't know but look if they are different...
how long do you wait untill you take away the combs? I use to wait no more than 1/2h. and I add the running buffer inmediatly. My boss use to clean the wells with a syringe, I don't because it's a waste of time (my point of view)
hello,
I just made 2 new gels again today and came and checked on them after 20 mins, and I've never seen anything like it!
On Both gels; the left hand side of the stack has completley disappeared. On the other, right hand side of the stack, the wells have formed okay!
Why on earth would this happen on both gels?!
It makes no sense I'm going to have a nervous breakdown.
and do you mix the mixture well? I use to invert the mixture several times...
I propose make everything new, buffers, APS, AA... for sure there's something wrong but, where?
good luck and don't desperate!!!!
thanks I will try inverting the mixture a few times to see what happens.. its the only thing i can think of really.
I havent really mixed too much before, as someone once said about introducing air into the mixture can make it shrink...
I think I have no choice!
I havent really mixed too much before, as someone once said about introducing air into the mixture can make it shrink...
I think I have no choice!
you should still always mix well.
do you have your gels polymerizing near an air vent or outside wall or radiator. it sounds as though your gels are in slightly different temperature zones when they polymerize differently and as though they are in a temperature gradient when they polymerized differently side-to-side.
I havent really mixed too much before, as someone once said about introducing air into the mixture can make it shrink...
I think I have no choice!
you should still always mix well.
do you have your gels polymerizing near an air vent or outside wall or radiator. it sounds as though your gels are in slightly different temperature zones when they polymerize differently and as though they are in a temperature gradient when they polymerized differently side-to-side.
I see. This time i did have them in the fume hood actually because someone said i should leave them in there beacuse of the acrylamide. Do you think that could have been it?
yes, it may be. with the air currents in the hood you may be getting uneven cooling of your gel, resulting in uneven polymerization. you would have to allow the gel to polymerize for extended periods of time (until it is complete).
there is no need to polymerize in the hood. once acrylamide is in solution there is no danger of fumes or airborne particles (unless you sneeze into the tube, flask or plates) from the acrylamide.