Agarose gel electrophoresis troubleshooting - (May/01/2009 )

i'd suggest two things:

1.if u're putting EtBR in the microwave-melted agarose in TBE/TAE..don't put it until the solution is cool!if u put EtBr when its boiling hot..it will degrade.

2.Try changing the loading dye?maybe it has too less glycerol?

Hi there!

I'm working on a new lab and i'm having a problem with my gel.

Why my DNA samples are running like this (see the figure)? The two lanes are 100 bp DNA ladder. All samples look the same. 1% Agarose gel, 80V 90 mA. I just don't have enough resolution with small fragments.



Brand new DNA ladder (promega)
Brand new agarose
Buffer TAE

Any ideas? Suggestions?

Thanks in advance!!!

1% agarose will not resolve 100 bp fragments well under any circumstances. Try 2% agarose, or (better) 3% Nusieve 3:1 agarose.

Did you run your gel long enough?

I use a 2.5% agarose and use a 100bp ladder. We use Seakem Agarose here and for better resolution, I would suggest using metaphor. Anyways, just increase the % of agarose to 2 or 3 % and you should be fine. Also, you should either stain with EtBr longer or add more EtBr when you make the gel. With little resolution and enough EtBr, the bands are blinding bright but yours are giving a weak signal.

Ameya

Thank you guys, I tried a new agarose, and the resuld was really better!

Thanks!

hi there,
I have a similar problem: I have nice bands of DNA and marker on top of the gel (the side where the wells are), but exactly at 1/2 of the gel, they disappear... So no bands there, at least the marker should be visible.
I use 0.5% TBE Buffer (pH about 7-8 according to pH paper), tried different loading dyes, and stain with texasred (tried 1/2 to 1.5 hours, with 10-20ul per 100ml buffer)...
Any suggestions why this happens?

thanks a lot

I used SYBR Green and nothing like that happens...

VebXperts Bpo

Ninana, the problem you are having with half of the gel unstained is caused by migration of the dye towards the negative electrode, while the DNA is migrating toward the positive electrode. This leaves the lower part of the gel unstained, since there is little dye left in that region. You can solve this problem by adding dye to the buffer tank in the positive electrode region, or by post-staining the gel.

Hi,
thanks for the answer. But I do post stain the gel, so the dye is everywhere... but:

problem solved:

It was the agarose. At least when I opened a new agarose, I suddenly got better gels. Attached is a picture of the old agarose (left) with a 1kb and 50bp marker, and the new agarose (right), with 1kb marker, my PCR products and a 50bp marker. I run the 2 gels together, so same conditions.
Does anyone know why the old agarose didn't work anymore? Maybe it got contaminated?