my colleague isolited RNA from tissue with TRIzol and phenol-chloroform extraction and I from cell in monolayer with RNeasy mini kit. Her protocol includes 2 min heating at 70°C after DNase purification and before checking on non-denaturatd agarose gel. Can somebody explain why she was using this step? I cannot see any differences between heated and non-heated samples on my gels.
The heat treatment is used to inactivate the Dnase. very typical.
The answer to why inactivate the Dnase is here:
DNase I treatment is clearly the best way to rid an RNA sample of contaminating DNA. However, some preparations of DNase may be contaminated with RNases, and the DNase must be completely inactivated prior to RT-PCR so that it doesn't degrade newly synthesized DNA.
Here is some background info (from the RNA company, Ambion, great little source for tech notes and products) ---> [Only registered users see links. ]
The Following User Says Thank You to danfive For This Useful Post: