Elution of a smear from acrylamide gel - Electrophoresis and gel elution (May/31/2007 )
Hi!!!! I'm trying to purify DNA from a polyacrylamide gel, in order to perform a PCR. THe sample is cDNA obtained from total RNA; so, it has some contamination from ribosomal RNA. So, in the gel, I obtained a smear with some very bright bands, that I suppose are from ribosomal RNA and I don't want them. I excise those bands, and I put the rest of the smear in 500ul of water (because I excised a lot of smear without bright bands). I incubated to 65C, and I added another 200ul because the gel didn't dissolve correctly. But I didn't obtain a good elution of the gel slices . What I can do to try to obtain the great yield possible from this preparation????? Thank you!!!!
sorry, but what was the reason for running a electrophoresis with cDNA?
you always will get a smear when performing cDNA synthesis with random primers. for me it makes no sense. for further pcr you directly could use the product of your reverse transcription.
i agree with moljul, u dont need to pass ur cDNA on gel b4 ur pcr, u choose the suitable primers (not random ones), then u do ur pcr directly and if u think that ur cDNA is contaminated with RNA, y dont u treat it with RNase?