Problem with agarose gel electrophoresis - (Dec/01/2008 )

QUOTE (mdfenko @ Nov 28 2008, 08:18 PM)

QUOTE (dr.dalia @ Nov 28 2008, 07:11 PM)

bands faint quickly before the marker separates well
so that to get good obvious bands, marker is not well separated yet & needs more time,
but if i left it for more time, bands faints !!

I don't know how to fix that. Can the problem be the agar type or what???

most likely caused by migration of ethidium bromide. it migrates through the gel in the opposite direction that the nucleic acids migrate.

you can include etbr in the electrode buffer or stain after running the gel.

i have bands but [b]they faint withen 20 min.[/b] on the gel !!

does ANYONE knows WHY? & HOW to fix that

Please help.
Thank you

-dr.dalia-

QUOTE (dr.dalia @ Dec 1 2008, 03:41 PM)

QUOTE (mdfenko @ Nov 28 2008, 08:18 PM)

QUOTE (dr.dalia @ Nov 28 2008, 07:11 PM)

i have bands but [b]they faint withen 20 min.[/b] on the gel !!

does ANYONE knows WHY? & HOW to fix that

Please help.
Thank you

Not sure what you are looking for. mdfenko already gave you the most likely cause. As you further run the DNA in gel, EtBr will migrate the opposite way and DNA bands will have less EtBr to intercalate with and hence less fluoresence. The way out is to add some EtBr in the opposite chamber buffer, or just restain-destain the gel with EtBr.

Did you mean that you see the bands under UV, then leave the gel outside and if you look again in 20 minutes, the bands have become relatively faint? If you have small DNA fragment, it will diffuse with time unless it is running, and so the bands will become diffuse and faint. Fluoresence will also decline with continued exposure to light/UV.

-cellcounter-

Are you looking at your bands all the time when running your gels? If so, UV degrades the DNA and photo-bleaching of the ethidium bromide occurs, meaning that you will no longer be able to see your bands.

-bob1-