Cleaning up RNA after double enzymatic reaction - (Sep/08/2008 )
Hey,
I have to do twice enzymatic reactions -exonuclease digestion of rRNA and poly-adenylation- on my RNA... the problem is, since my RNA comes from a very dirty source I do spin column purification before processing... then another spin column purification (RNeasy) after digestion and another after poly-adenylation... then I have so little material left, and this RNA is really precious, I really don't have any luxury to lose material... does anyone have an idea how to clean up RNA after enzymatic reactions without losing much stuff?
thanks!
I have to do twice enzymatic reactions -exonuclease digestion of rRNA and poly-adenylation- on my RNA... the problem is, since my RNA comes from a very dirty source I do spin column purification before processing... then another spin column purification (RNeasy) after digestion and another after poly-adenylation... then I have so little material left, and this RNA is really precious, I really don't have any luxury to lose material... does anyone have an idea how to clean up RNA after enzymatic reactions without losing much stuff?
thanks!
I always think traditional methods are more reliable though it's time-consuming. If you have troubles using columns, try Phenol extraction and EtOH ppt. Actually I just have experience with EtOH ppt with RNA samples, and it always resulted in good recovery. I heard phenol extraction with RNA samples is little bit different from the one with DNA. Good luck anyway.
You can often switch buffers and work a second reaction without purification. Determine how similar the buffers for the two reactions are. You may have to inactivate the first enzyme, but this can often be done by heat killing.
Hhmmm... thank you both.. I've tried ph:chlorofom already, but I didn't really get much yield, especially since the concentration of mRNA is too low... Heat inactivation is a good idea, but do you think RNA would be till ok after heating to 90 degrees?