Adding ethidium directly to samples prior to loading - (Apr/05/2008 )

Hi everyone,

I've read somewhere about adding ethidium directly to the samples to be run on gels prior to running. I will be using acrylamide gels.

Has anyone done this? I am trying to minimize background while having nice sharp bands for microarray RT-PCR validations.

One protocol I saw said to use 0.4ug/mL in the sample, wait 5 minutes, and then load and run on gel as usual.

Does this result with streaky gels? In theory it should work....since EtBr runs opposite direction of DNA, I would think any unincorporated EtBr would just float up into the buffer in the upper chamber. This should leave just the EtBr-treated bands migrating into the gel.

Thanks!

Thanks! Do you know what concentration you used?

I tried it today with 0.4ug/mL EtBr and waited 5 minutes before loading.

Nothing showed up except the two largest bands (1kb and 1.2kb) on the ladder that showed up very faintly.

I guess I should raise the amount of EtBr?

Yes, I would increase the amount of Ethidium. Some protocols say to use a final concentration of ~10µg/µl. I have used less than this, but usually had to use the "integrate" feature on the transillumination software to visualize well. (Increase the exposure time). Sometimes you can play with the exposure time or other settings on your software to get better visualization without having to add too much EtBr.
When you're adding EtBr directly to your samples, do make sure to add a uniform amount to all of them (including the ladder), since EtBr effects nucleic acid migration rates through the gel.
Hope this helps..

Thanks so much! I'm assuming you mean 10ug/mL and not 10ug/uL since most EtBr stocks come at a concentration of 10ug/uL.

I'll try it on Monday!

Oops -- sorry! Yes, I meant 10µg/ml...
Great -- good luck!

Hmm...so I tried it today and it still didn't work =( Once again only the two largest bands of the ladder showed up (and very faintly at that).

I put 30uL of 10ug/uL into 10mL of loading dye. I then added 5uL of this to 10uL of PCR so the loading dye would be diluted to 1/3...so total was 10ug/mL in the sample....

Maybe there's something about acrylamide that makes the intercalated ethidium come off the DNA and migrate away. =/

Hmm...
Is your background too high if you stain/destain the whole gel? (Sorry, I know the whole point was to get away from that...)
10µg/ml of EtBr in the samples should have been plenty. Honestly, I don't know why it didn't work. Sorry about that...

No problem, thanks so much for your help. But as we all know, things that should work in the lab often don't =)

The background is a tad high, but I'll just have to live with it.

Thanks again!