10X MOPS running buffer (50 ml):
0.4 M MOPS, pH 7.0 (1M pH7 stock - 20 ml)
0.1 M sodium acetate (3M stock - 1.67 ml)
0.01 M EDTA (0.1 M stock - 5 ml)
DEPC'd H2O 23.33 ml
(Note: the buffer ends up about pH 5, is that a problem?)
10ml of 10xMOPS buffer,
18 ml 37% formaldehyde
1X MOPS running buffer (30ml 10x MOPS buffer made above & 270 ml DEPC'd H2O)
formaldehyde 45 μl
formamide 45 μl
10X MOPS buffer 10 μl
EtBr (10 mg/ml) 3.5 μl
0.1 M EDTA (pH 7.5) 1.5 μl
bromophenol blue dye (in 50% glycerol) 8 μl
Add10–15μl of RNA loading solutionto 1–2μg of RNA
Heat at 70°C for 10–15 min., cool on ice 1 min, then load on gel.
As a habit, I added EtBr to the gel, but I then realized that this lights up the gel and I can't see any bands. So, I ran another gel, without the EtBr, but all I could see was a large bolus of the EtBr running towards the anode (as it EtBr does), but, to my surprise, I did not see any bands of DNA running towards the cathode at all (at this point I am only running a DNA ladder so that I wouldn't waste RNA). I checked the DNA ladder on a regular agarose gel (with EtBr in it) and it worked perfectly. I also checked it on a MOPS buffer-only gel (no formaldehyde), with EtBr in the gel, also worked well. The only problem seems to be when I add formaldehyde to the gel. Any ideas? I talked to at least a couple "RNA experts" form our suppliers, but received no clues.
Edited by DNAgeek, 03 September 2010 - 06:08 PM.