Troubleshoot SB buffer electrophoresis problem - (Nov/23/2007 )

Inspired by good reports about sodium borate buffer (SB) and how you can run your gels faster and still get decent resolution I decided to give it a whirl.

I made three gels, one with our standard 0.5X TBE and two with 1X SB. I loaded the tanks with buffer to match the gels and all the gels with the same DNA samples. I ran the TBE gel and one of the SB gels at 150V as is standard practice for my lab. The other SB gel I tried running at 200V.

Well, my TBE gel ran the same as it usually does - getting a bit warm and taking five hours (it's a high percentage gel).
The SB gel at 150V got considerably warmer (i.e. it curled up and slightly melted at the end) than the TBE gel and the DNA only migrated half the distance of the DNA on the TBE gel after the same five hours.
The SB gel at 200V was more or less the same as the one at 150V but just more melted etc.

I suspect I may need to reduce the concentration of the SB down to 0.5X to get it to run cooler but why didn't the DNA migrate so far?

Any thoughts?

Thanks
MTO

-MTO-

SB buffer has less electrolytes in it, the ions conducting the electricity get depleted faster and hence the gel didn't run as far and the resistance went up producing heat and melting your gel.

-bob1-

QUOTE (bob1 @ Nov 26 2007, 12:14 AM)

SB buffer has less electrolytes in it, the ions conducting the electricity get depleted faster and hence the gel didn't run as far and the resistance went up producing heat and melting your gel.

Thanks bob1.

So would I need to use more concentrated SB to counter the electrolye depletion or should I just give up and use TBE with really long running times?

-MTO-

check this link for more on using sodium borate:

sodium boric acid as tae and tbe replacement

and compare with your formulation.

-mdfenko-

QUOTE (mdfenko @ Nov 26 2007, 08:21 PM)

check this link for more on using sodium borate:

sodium boric acid as tae and tbe replacement

and compare with your formulation.

Thanks mdfenko. That's the thread I'd been looking at before.

I went with the recipe given in post 15 of that thread:

8g NaOH
45g Boric Acid
ad 1l H2O
(makes a 20x solution)

mainly because I could see sodium hydroxide and boric acid on the shelf.

-MTO-

maybe you should try the formula in post #2 (don't forget to use borax) or the formula in post #11.

the formula that you used is a 20x solution. if not diluted properly could cause salt effects (eg-heating).

-mdfenko-

QUOTE (mdfenko @ Nov 27 2007, 09:54 PM)

The formula that you used is a 20x solution. if not diluted properly could cause salt effects (eg-heating).

I did dilute to 1x but looking at Bob1's reply I wonder should I use more concentrated SB to counter the electrolye depletion?

I'll try borax next!

-MTO-