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RNA cleanup methods (WITHOUT USING KITS!!) - protocol for removing phenol contamination (May/12/2009 )

Hi,
I've tralled the net for a protocol for cleaning up RNA contaminated with phenol. i've seen various posts on this website but no definitive protocol. someone refered to re-precipitating the rna with chloroform & then ethanol.

HAS ANYONE GOT A PROTOCOL THAT I COULD USE!!!!!

thanks x

-Gillian-

- add 1 volume of chloroform and vortex 3-5 min
- centrifuge for 5 min at 10.000 g
- pipette the upper phase in a new tube
- add 3 volumes ice-cold ethanol, 1/10 volume of 3 M NA-Acetat pH 4.8 (or ammonium-acetate) and 1/100 volume glycogen (optional)
- incubate at -20C for at least 1 hour (better over night)
- centrifuge for 30-60 min at 4 and 10.000 g
- discard supernatant and add 1 ml 70% ethanol
- centrifuge for 15-30 min at 4 and 10.000 g
- discard supernatant and dry the pellet (speed vac or on the bench)
- resuspend the pellet in A. dest or TE

Not much different from cleaning DNA...

-mastermi-

<_< Thank you! i wasn't expecting a response so quickly.

One question...what is A. dest? and is it okay if i resuspend in RNAse free water?

-Gillian-

A. dest = Aqua destillata

You can use any RNase-free water.

Indeed I use autoclaved MilliQ-water...

-mastermi-

Hi,

How much RNA (%) you can precipitate? I did similar protocol and I could recover 10% of my RNA. Which ratio did you obtain?

Thanks!

mastermi on May 12 2009, 09:17 PM said:

- add 1 volume of chloroform and vortex 3-5 min
- centrifuge for 5 min at 10.000 g
- pipette the upper phase in a new tube
- add 3 volumes ice-cold ethanol, 1/10 volume of 3 M NA-Acetat pH 4.8 (or ammonium-acetate) and 1/100 volume glycogen (optional)
- incubate at -20C for at least 1 hour (better over night)
- centrifuge for 30-60 min at 4 and 10.000 g
- discard supernatant and add 1 ml 70% ethanol
- centrifuge for 15-30 min at 4 and 10.000 g
- discard supernatant and dry the pellet (speed vac or on the bench)
- resuspend the pellet in A. dest or TE

Not much different from cleaning DNA...

-sus12-

sus12 on May 20 2009, 11:51 AM said:

Hi,

How much RNA (%) you can precipitate? I did similar protocol and I could recover 10% of my RNA. Which ratio did you obtain?

Thanks!



I always used this protocol directly after RNA isolation. After isolating and cleaning my RNA I get about 40 g RNA out of 1 ml exponential bacterial culture.
But I never knew how much RNA I had before phenol-chloroform-extraction...

-mastermi-