DNA extraction using RNAse (help needed) - (Apr/21/2005 )
I reckon yesterday I posted this in the wrong place. So now I'm doing it again (hopefully in the right place).
Well, it happens that we want to proceed to a DNA extraction from blood cells using the phenol-chloroform method including a RNAse step somewhere in the middle of the process. We don't know the optimum conditions (time, temperature...) for RNAse use and that is why we're asking for help.
All answers are welcome.
your protoco;l may have a lysis step followed by phenol chloroform assay?
i use to do an RNAse treatment for 15' at 37° followed by proteinaseK treatment (15' 37°) just before the phenol chlo step. By assuming the conditions won't be good for enzymes, i can only suggest to pellet DNA after the first phenolchlo resuspend it in 500µl TE buffer and do the RNase step.
For 500µl i use 1.5µl RNase (10mg/ml) followed by 2.5µl of proteinase K 20mg/ml)
hope that helps.