RNA precipitation - How long should I precipitate my RNA? (Jul/08/2005 )
I just have a question regarding RNA precipitation:
I have to do a lot of RNA extraction from frog tissue and after DNAse treatment the protocol asks to precipitate with Ethanol/NaAce o/n at -20C (or minimum 1 hour).
I would like to know how shorter times at -20 affect RNA recovery? What the "speed" od precipitation of RNA? Is there any other method that might be better for faster precipitation?!
when you isolate RNA from an aquous solution using a guanidine thiocianate method you precipitate the RNA with the isopropanol (1 volume) at room temperature for 10 minutes. I got good yields (5-10 ug from 1 million cells)
Hi, after you have isolated your RNA and resuspended the pellet use Ambions DNA free kit, you simple add some buffer to your RNA then the DNAse incubate @ 37 for 30 min or so then add some DNAse inactivation reagent this binds the DNAse you centrifuge and remove the supernatant (DNA free RNA) it is far easier using precipitation techniques and will help reduce losses.
I use Trizol for prokaryotes but there is a Trizol LS ? for tissue, basically lyse or bead beat cell, bead beating is best as lysing causes stress - stress related proteins (mRNA), after trizol add some chloroform to separate layers, centrifuge remove supernatant to fresh tube and ptt RNA using isopropanol @ RT for 15min, then wash in 70% EtOH and air dry for 15min, resus pellet in H2O and DNA FREE treat.