Looking for perfect acrylamide gel EtBr stain - (Mar/29/2008 )

Hi everyone,

I have a CBS Scientific Triple Wide acrylamide gel rig (the plates are 33x10cm). I post-stain the gel on one side of the plate (it is UV-transparent glass).

Does someone have a good EtBr concentration and stain/destain times to get great images?

When I use normal 10x10cm acrylamide gels, I stain for about 3 minutes (1ug/mL) and then destain for 5 and the images are great....pure black background with nice white sharp bands.

However, I tried 5 minutes EtBr stain (1ug/mL) and 10 minute destain and it was still sort of high background.

Would it be better to increase the EtBr concentration? Or increase/decrease stain time? I am looking for bright bands on a black background instead of a greyish background.

Thanks!

if it worked perfectly at 3 minutes stain, 5 minutes destain then why did you change the timing? as long as the gel is the same thickness as the smaller gel (which i assume because you did not mention a change in thickness, only width) then the timing should be the same.

Thanks for your reply. The difference is that I usually remove the gel from the plate for the 10x10cm small gel. For the 33x10cm wide gel, the gel is left on one of the glass plates.

So I guess it changes things that only one side of the gel is exposed to the ethidium.

can you remove the gel from the plate?

is the gel fixed before staining?

prolonged exposure to the stain and destaining solutions may be leaching the sample from the gel if it is not fixed. you may still want to use the shorter times of the small gel.

The gel is too fragile to remove for staining. It is the same width as the small gel, but it's sheer width makes it very difficult to handle.

And no, it is not fixed first.

I'm just not sure if I should increase the concentration of EtBr in the post-stain solution because that would also increase background.

Guess the only thing to do is to give it a try and see. =)