RNA precipitation - (Mar/09/2002 )
i would like to precipitate again my RNA extracted from veray little tissu.
How can i make
what is your tissue?
rna ppt is very similar to dna ppt.
salt and ethanol are used and nucleic acid is allowed to ppt and spin and resuspend in a smaller volume.
look on current protocls or the web for RNA ppt methods. but like i said it's similar to dna ppt.
If you try Sean's suggestion, you may also want to use glycogen to act as a carrier in the alcohol to help get the most RNA out. It works pretty well and won't affect your RT-PCR.
So RNA co-precipitates with DNA?
i've always used phenol chloroform precipitation.
during phenol extraction, the precipitated proteins collect at an interphase between the aqueous and organic layers. for the purification of DNA, the use of phenol at pH above 7.0 is desirable in order to collect DNA in the upper aqueous layer of a phenol extraction (phenol saturated, pH8). At lower pH (4.5), DNA and proteins are collected at the interphase although RNA can be collected in the aqueous phase (phenol saturated, pH4.5).
if you're going to do RT PCR, remember you have to turn it to cDNA first.
I used Ambion ribopure kit to extract RNA and replace chloroform with BCP, I think I have RNAse contamination in th prep. since after my samples was stored at -80C fro a week, they are degraded. anyone with similar experience or suggestion?
Yes, both are very similar from a chemical point of view. If you can get them separated first, then you're fine, but if you precipitate a mixture, both will get pelleted.