EMSA Help me please!! - (Aug/09/2013 )
I'm working on EMSAs and recently tried switching over my gel format to a large gel (16.5 cm x 28.5 cm) instead of a mini gel to get better separation without losing my free biotin labeled DNA. This is the first time I've tried using the large gel and am having trouble figuring out appropriate gel settings (ie volts and time). First off, everything if being run at 4 degrees and the gel is 4% polyacrylamide in .5X TBE.
I originally tried running the gel at a constant 150V for about 8 or 9 hours but stopped because I could see I was starting to get a smiley face effect. So, I thought maybe if I ran it really slow I could avoid that and instead ran it at a constant 65V for 12hours. I didn't see any curvature in the samples from looking at the loading buffer so I decided to increase the voltage to 85V and ran that for 3.5hours. Now it just looks like my samples are almosrt dispersing by looking at the loading buffer (it usually looks like 2 slightly different colored blue bands that are pretty compact - around the width of a key) and now it looks like the blue bands are about the height of a dime. So I'm not sure if the gel is running appropriately.
Also, I would like any input regarding transferring the gel. Could I get away with transferring it at the same volatge as a mini gel just running it longer? Or would I need to increase voltage as well?
Any suggestions/help is greatly appreciated!!
I am so sorry if this response is late for you. Put I am using the following EMSA protocol and I hope that it may be helpful for you.
I use 6.5% non-denaturing polyacrylamide gel and run it in 0.5x TBE. Before you load your sample, please run the gel in pre-chilled 0.5X TBE buffer for 10 minat 120V.
Load your sample, then put the gel tank on ice water in ice box or run in 4C refrigerator at 100V til th edye reaches 1 inch from the bottom of the gel (approximate time 50-60 min).