Please help- running conditions - (Jul/10/2010 )

Hello,
i am running some PAGE gels and I think I am having some problems in my PAGE running.. could you please inform me of the conditions that you use (voltage?, mA?, time? you think is enough for separating 107, 82, 25bp in 12% Page) you treat which parameter as your constant V?/ mA?.. I have been trying to trace the migration with the loading dye but this seem to be very difficult task, the dye is v. faint (I am adding 1ul of 6x loading dye(bromophenol blue, xylene cyanol) to 20-25ul of sample ,is this fine?)..

Fhannan on Jul 10 2010, 03:47 PM said:

Hi there

It was not clear from your question whether you are referring to denaturing PAGE gels. If that is the case, I normally run 8% UREA-PAGE gels to resolve 40 bp fragment at 20 - 30W constant which will be more or less equal to 900 - 1000 v or 20 - 30 mA. I guess it would be better to run at constant W. Obviously, you are loading too less dye to the sample, your final concentration of the dye should be 1x in the sample so if you have 6X loading dye you should load at least 4 ul for 20 ul sample. Hope this would help.

Fhannan on Jul 10 2010, 04:47 PM said:

normally we run 12% PAGE at constant voltage i.e., 120V for first 5-10 min and then 200V for around 40 mins. To see how much time your samples need to complete/migrate, you can run a pre-stained ladder in beginning, although usually the dye-front helps to see that. i normally use 50% loading buffer (laemmli sample buffer) for my samples but again you need to try out according to your sample type and amount.