Agarose: DNA/dye mixture doesn't stay in the gel? - Thanks for any and all help received! =] (Jul/15/2009 )
xCj425x on Jul 17 2009, 12:07 AM said:
I'll make sure about the ethanol next time I extract DNA.
But the gel thickness issue definitely also makes sense regarding why the mixture would float out of the gel itself, instead of just the wells. I know that the apparatus is level, but the middle of my gel always is lower than the wells itself.
Also, I could definitely be using overheated gel. The problem lies in the fact that I don't exactly know how hot is "too hot"
I always just assumed that as long as it's melted, it's okay, so I usually end up heating it up to when I know for sure it's hot enough. Whoops.
Thanks so much for all the responses!
I'll post again after applying the fixes mentioned above.
As for how hot is hot, it should be comfortable enough for you to hold in your hands.
What I always do is to boil about 500mL or 200mL of 1% agarose, and then keep it in a 55degcel incubator. I will then take the gel from the incubator and pour the necessary amount whenever I need to, before putting the rest back into the incubator.
I have had this happen when I try to make thin gels to save agarose. Even if you do have a gel of even thickness there will be a larger portion around the wells...you can thank adhesion for this. I haven't had any problems visualizing my bands on these gels either.
Try making a thicker gel or loading less.
Yeah, so my problems are basically fixed.
It was a combination of the fact that I didn't get rid of the ethanol properly.
(After removing ethanol completely before suspending the DNA, the DNA wouldn't float out of well anymore when used in electrophoresis)
Also, georgiadave brings up a great point.
Adhesion makes it so that the gel is slightly thicker around your wells, and so when you run the gel, the DNA comes leaking out when it runs out of gel to run through, and starts pouring out of the gel itself.
Making thicker definitely helped, but as phage mentioned, putting the comb in first helps deal with the adhesion issue.
I noticed that using slightly more gel, but also assembling the comb first, resulted in a completely flat gel surface.
Thanks so much for all your help everyone!