agarose gel

[hara]

i usually make my agarose gel this way:
boil agarose powder in TBE 0.5X in a microwave oven then i let it cool a little and put ethidium bromide stir well and put it in the track to polymerize...
recently i heard that some people put ethidium bromide in the TBE solution before making the gel
has anybody more information about this way?
thanks

[SororSaudade]

i've never heard of that, but how do they melt the agarose? Aren't ethidium bromide vapours a little bit dangerous?
I usually do it the way you described (but with TBE 1x) and I know that some people run the gel without ethidium bromide and after the run place it in an ethidium bromide solution.

[sachin]

Then How it get inter calated? After running gel?

[SororSaudade]

I don't know how because i've never done it

just to add something to my previous answer... I belive ethidium bromide is heat degradable and I can't see any advantage in adding it to the TBE solution, but I might be missing something...

[canalon]

Definitely adding BET should be done after boiling and not before. I know some people prepare more gel than necessary, add the BET and then reheat when needed for the next gel. But this is definitely dangerous.

Since I am a firm believer of "the least BET contamination, the better I a feel" I do stain my gels after running them (10 minutes in an TBE/BET solution, rinsing in water or TBE for 10 more minutes et voila) so I limit contamination to 2 plastic boxes, and I keep all gel boxes, pouring apparatus and buffers clean.

[LilKim]

Definitely adding BET should be done after boiling and not before. I know some people prepare more gel than necessary, add the BET and then reheat when needed for the next gel. But this is definitely dangerous.

Since I am a firm believer of "the least BET contamination, the better I a feel" I do stain my gels after running them (10 minutes in an TBE/BET solution, rinsing in water or TBE for 10 more minutes et voila) so I limit contamination to 2 plastic boxes, and I keep all gel boxes, pouring apparatus and buffers clean.

I wish my lab-folk did that. They think that i'm paranoid because i don't like to add ET to my hot agarose soln. and re-heat it.

Also, beside releasing vapors.. is it possible that re-heating the agarose+ET solution would cause the ET itself to 'break-down'? (that's what i've heard, but don't know if it's true)

Oh.. and in my lab we make .8% agarose gels using TAE.

[SororSaudade]

i've always heard that EtBr is heat-labile (its altered by heat).
i also think that it doesn't really matter if you use TBE or TAE... at least for DNA electrophoresis.

[canalon]

TAE, TBE, not much differences. Percentage of Agarose depends of what you are looking for.

And yes BET is heat labile, but not that much so it is OK if it boils once, much less so if you keep it a long time in a warm environment (I knew someone who kept his melted agarose at 60C in an oven with BET, once he left on holiday for 2 weeks, and when he came back his agarose was rusty red yuck)