RNA extraction from sludge - (Nov/10/2005 )
Hi everybody !
I'm trying to extract RNA from sludge samples coming from different wastewater treatment plants and I have lot of troubles. I'm using a classical protocol with a mechanical lysis with beads and a beadbeater and hot phenol and then I do several phenol/chloroform extraction (up to 5 in some cases) and finally 2 chloroform extraction to remove phenol. Then I precipitate with ethanol at -80°C. After precipitation, I wash the pellet with ethanol 80 % and dry it before dissolution in DEPC-treated water.
The problem is I never get the same results from one experiment to another... Sometimes I have a final ratio 260/280 of 1.7 but sometimes it is more about 1.3 and in that case I can't see anything on agarose gel.
I also have a lot of DNA hard to digest (about 10 µL of DNase I required for a 50 µL sample !!). Do you have any solution to limit the presence of DNA ?
I tried to lower the pH to 4 (instead of 5 initially) with no effect on the DNA quantity. I don't know really the effect of pH cause in several RNA extraction kit I noticed that the pH of buffers are at 8 even for RNA extraction. Does it matter really ?
Second problem : my samples have been frozen before any manipulation. So I have for each sample 50 mL of frozen sludge. To treat these samples i need to thaw them and then centrifuge, wash, resuspend, aliquot in smaller tubes and then extract... The problem is that I think the thawing phase will degrade the major party of my RNA. Do you have any suggestion to prevent the degradation during thawing ? Maybe phenol could protect the RNA from RNase ?
Thanks a lot for your answers
what organism you are looking for, bactteria or viruses?
Your RNA purity is low, how about the RNA concentration?
I'm looking for bacteria to do dot-blot experiment.