LiCl riboprobe precipitation - (Feb/08/2008 )
Hello! I have this protocol to purify riboprobes. is it ok? Thank you a lot.
- 0.1 Vol LiCl
- 2.5-3.0 Vol di Et-OH iced (70% or 100%????)
- 80°C for 30’ or –20°C O/N
- 13000 x g 15’
- discard supernatant
- wash pellet with 100 ul di Et-OH 70°iced
- 13000 x g 5’
- dry pellet (owen 45°C)
- eluite pellet in 20ul (50ul H2O)
- kept at –80°C
Not so experienced that I can say if your protocol is good or not. But I guess that the first ethanol has to be 100% (above 96%), else the RNA will not precipitate. Or maybe it will because of the LiCl.
I have used the following protocol for precipitation of RNA transcript (may not be efficient to precipitate RNA smaller than 300bp).
- Add 30 ul of LiCl precipitation solution (7,5 M LiCl, 50 mM EDTA) and 30 ul of nuclease free water.
- Mix. Chill for 30 minutes or longer (-20)
- centrifuge at 4 degrees, max speed, 15 minutes to pellet RNA
- Remove the supernatant, Wash with 1 ml 70% ethanol. Centrifuge again.
- Remove the ethanol and resuspend in appropriate solution (I used DEPC-water).