I did in vitro transcription reaction by Sp6 polyermase synthesizing DNP labeled RNA from a digested and pheno/chloroform extracted, ETOH precitipated plasmid DNA template. I put at 37C for 2.5 hours. I used about 1.5ug DNA template. However, after the reaction when I run the gel, the synthesized RNA is dimmer than the DNA band. According to my protocol, the synthesized RNA should be 10X brighter than DNA template. I don't know why. My DNA has a nice peak at 260, and 260/280 ratio is 1.79, which is nice. However, I have a very low 260/230 ratio, which is 1.56. Could anyone give me some suggestions?
Hi Michaela, Thanks so much for your reply. I saw some protocols that even precipitate at -20C for overnight. I think it would be fine to precipitate longer since we still use 70% ethanol to wash away. For phenol/chloroform, I did use an old one, which is like one year old. However, I didn't see any gunk at the bottom. I suppose it is clear?
Thank you Michaela. Actually I washed twice with 70% ETOH after 100% ETOH. I think I removed most of salt, and my only salt added is NaAc. I found the peak of it is at 270. However, I don't see a peak shift to 270 in my purified DNA. Normally I cannot see DNA pallet, but I added glycogen to let me see the pallet. The pallet is white and small and at the edge of the bottom of the tip. For the phenol/chloroform, Actually I used pheno/chor/isoamyl mix solution followed by twice of choloform. Would that be difference? You said all components warm to room temperature. I put polymerase and labeling mix on ice, and before reaction, I heat DNA to 55C and cool on ice. Would it influence?
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