Hot Phenol and RNA shearing? - (Mar/11/2008 )

I understand that when doing phenol:chloroform extractions for genomic DNA it is not recommended to vortex the sample as it may cause shearing of the DNA. However, when doing an acid phenol:chloroform extraction for total RNA, would vortexing the sample cause shearing of RNA?

Also, when extracting RNA using hot phenol, how long does everyone incubate the sample at 60-65C for? Protocols I've referred to range from 5 - 60 mins and I've been told by another member of the lab that longer incubation resulted in higher RNA yields. However, I feel that such long periods of incubation would damage the RNA.

Sorry for all the questions and thank you in advance.

Hey,

Genomic DNA may shear when vortexing because it is high molecular weight. This is not the case for RNA. I never used hot phenol for extraction, sorry:(

Hi !

I usually perform 2 hot phenol shocks of 2 min each at 60°C with mechanical lysis in beetween...
Don't be afraid to vortex, RNA won't be cut cause it's a smaller molecule than genomic DNA.

Bye