Ethidium bromide in formaldehyde gel

[tannadurai]

i use MOPS and formaldehyde gel to separate RNA i add EtBr in sample buffer. at begining above the well etbr expression is present i.e etbr moves opposite direction. at end of the gel it disappears may be run in to tank. whats wrong with this hw can i rectify this..

[nbaras]

I am having the same problem. I suspect we may be using too large a concentration of EtBr and the excess ethidium (positively charged ion in solution) runs towards the anode.

What concentration of EtBr are you using? "Molecular Cloning" recommends as little as 0.1 mcg/mL when using EtBr in loading buffer. I've used as much as 10mcg/mL and am testing the text's quantity today.

-Noah

[khalsakh]

I have ben using MOPs gel since 10 years ago and I used to get very nice separated 2 subunits. The image of seeing a high intensity EthBr above the wells is usually due to DNA contamination in the sample.

[mmorgan]

Originally Posted by tannadurai

i use MOPS and formaldehyde gel to separate RNA i add EtBr in sample buffer. at begining above the well etbr expression is present i.e etbr moves opposite direction. at end of the gel it disappears may be run in to tank. whats wrong with this hw can i rectify this..

Nothing is wrong with what you're doing. You should put EtBr into the sample loading buffer and it will run towards the cathode (opposite direction of RNA/DNA). You should still be able to see the rRNA bands under UV because some of the EtBr will intercalate and run into the gel.