I want to ask about the RNAse treatment for the cell cycle. What should be the ideal conditions during RNAse treatment i.e. Incubation period, temperature, time or either it should be placed in 5% CO2 incubator or not?
RNAse A is a very stable enzyme. The incubation period varies depending on the protocol, the cell number and your cell line RNA content(Many cells produce a lot of RNA, others not). Usually, the incubation period is 30 min at RT or 15 min at 37 C.
Good luck!
I use 50 µl of 100 µg/ml stock of RNase A. Regarding cell number , in the range of 5E5-1E6 works best for me.
Correct me if I am wrong, but the first step of the preparation is after cell washing an Ethanol-fixation, then Ethanol removal, PBS washing, then RNAse A treatment - at least in a Propdium Iodide based cell cycle protocol.
As soon as cells are fixed in Ethanol, they are dead - and why should they need 5% CO2 then?
Hi Sania,
I am agree with Daniela. After the cell fixation with ethanol, you don't need any CO2 incubator.
But for RNase A activity you need incubation at 37 C. In our protocol we always prepare a mix solution including PI + RNase A + Triton-X100 in PBS (final concentrations 50ug/ml, 0.1 mg/ml, 0.05% respectively). We use 1x10E06 cells per conditions/tubes and stained the cells with 500 ul of this mix solution at 37 C for 40 minutes at dark. It's worked fine for us so far. I hope it will work for you too.
Good Luck
Cigdem
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