electrophoresis buffer become very hot - (May/08/2007 )
recently i observed that when i perform agarose gel electrophoresis my electrophoresis buffer becomes very hot within 1 hour at 50 or 100v. i am afraid my gel will melt if i run for 1.5 hour or more
i can't understand what would be the reason
Too much salt will make it hotter. Perhaps your running buffer is too concentrated? Did you mistake a 50x solution to be 20x?
prepare fresh buffer and use more of it.
What buffer are you using??
We use 1x TAE most of our AGE, and then for things like RFLP which we run overnight (up to 20 hours) we use 0.5x TBE. The buffer does get warm, but not enough to do any damage to the gel.
I like to change the buffer at least once during these long ones though
i use 1 X TAE buffer
it is always made up of 10 L volume for common use. when it finish, then the person who want to use the buffer he/she make it from x50 stock.
for 10L volume we add 200ml of x50 TAE to make x1 TAE
i donno who is the last person, who made this buffer, i will ask other about this problem, whether they also face the same thing or not
and one more thing i found my gel is moving in gel plate at the time of electrophoresis......i stopped the machine and set the gel and start again and after 5 mins it start to move againnnnnnn.
what should i do to solve this problem?
one of my labmate tell me today that there was a lot of buffer in the tank when i electrophoresis my sample for this reason my gel was moved
is it right??
well if there is lot of buffer the gel tend to have difficulties to reach the bottom of the tank. Hence you need to put it yourself at bottom and wait few seconds to stabilizes. In case of very low gel percentage, myabe the gel density will be low enough to allow partial floating of the gel....
In my lab we run in 0.5X TAE. Maybe 1X is a reason for too high temperature?
For an electrophoresis you can use either 0.5X or 1x buffer the important thing is that must use the same for the gel and the tank. Check the power supply settings, it happen to me once and was that somebody change the settings and I did't check and trust and well got a boiling buffer with a melt gel and obviusly a cooked DNA.
well i performed gel electrophoresis yesterday with reduce amount of buffer in the tank and it wasnt move and the temp of the buffer was reasonable after finish the electrophoresis
usually we use 1X TAE buffer in tank and also same buffer for making gel
If the gel moves it's just because it got hot and started to soften and loosen from the tray. It's just a symptom of the same thing.