mRNA isolation without DNA!
watchcity at my-deja.com
Tue Feb 8 21:39:03 EST 2000
Hello Csaba, Have you tried the RNase-free DNase that comes with the rna
transcription kits? You could do an intact nucleus extraction, and chew any
remaining contaminants with DNase in an Rnase inhibiting (RNasin or some
such) environment. The downside is the cost. The DNase works well for
transcription synthesis. Cheers, Warren
In article <389fdefe at redeye.it.ki.se>,
"Csaba Kiss" <csakis at ki.se> wrote:
> Hi guys!
> I would like to do subtractive hybridization on cDNA. I want to isolate
> mRNA, which is free from even traces of DNA. The acidic phenol method is the
> generally used method to isolate total RNA, and it is based on the fact that
> the DNA will remain in the phenolic phase if the phenol's pH is acidic (ph
> 5.0 ?). Then people do mRNA separation on oligo-T beads or columns. I have
> a feeling that these separations do not get rid of your residual DNA
> totally. There is another way of isolating cytoplasmic RNA. You can gently
> lyse the cells in a way that the nuclei remain intact. You pellet down the
> nuclei, and work with the supernatant. This way you got rid of all the
> genomic DNA. However, in such a mild lysis buffer there is no Rnase
> inhibitor. How would I prevent my mRNA being degraded by the endogenous
> Rnase? Dynal recommends to work quickly on ice. I have a gut feeling that
> this does not eliminate RNA degradation. What do you think?
> csakis at ki.se
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