Im trying RNA prep for QPCR using the qiagen RNeasy kit. Im pretty sure that I was really really careful while extracting RNA. I added beta-mercaptoethanol to the lysis buffer, homogenized the lysate and froze it at -70. I did the prep after a week and I was pretty careful while doing it, so I doubt I made any mistake there. I didn't have enough RNAse free water so I pretty much used old TAE made in our normal distilled water. Since I was really anxious to see how it went, I used the DNA loading dye and DNA ladder because we didn't have the RNA versions. Heated the samples at 60 C for 5 mins and ran the gel at 130V (the electrophoresis tank is quite big, so I don't think the V/cm wouldve gone too high).
The RNA was degraded with a smear at 1.5-0.5Kb. I just wanted to see what happened if I ran the gel for another 40 mins. Interestingly after 40 mins of running, the ladder was still there (just separated more) but the RNA lanes were empty!!! I couldn't see the smear I was seeing before despite the equivalent ladder being right in the middle. I think it ran off. But does this mean that RNA got degraded DURING electrophoresis (became progressively smaller with time and ran off)? Do I need to add DEPC to the TAE? I got some RNA zap to clean the tank. But, what do I do with TAE. Can distilled water have RNAse?
Edited by vegeta, 12 July 2011 - 11:28 PM.