RNase treatment - (Jun/28/2006 )

i want to check the insert by RE digestion so i will just do sol1, sol2 and sol3 no phenol extraction etc.....but i want to treat with RNase....so when do I add it and how? like i ethanol ppt then dilute in TE then add RNase incubate and ethanol ppt again? ...or no ethanol pptation after RNase is needed? thank you for your help....

Your RNAse is not supposed to be contaminated with DNAse (otherwise you couldn't use it) and to contain a lot of salt, therefore you don't need to ppt again

hi
after sol 1, 2 and 3, i pellet DNA and RNA and resuspend in 10mM Tris pH 8.5 and RNase.
I let incubate 15' at 37° and then pick a sample for restriction digestion without removing RNase.

After Ethanol ppt., we used to add RNAse sol. to dissolve the pellet and use the solution directly for restriction digest.

thanx a lot everyone!

you can add RNase directly to your soln I
unfortunately I am away from the lab for a bit and don't have the concentration with me...but that is also how they do it for qiagen's DNA column prep-kits in their PI buffer

works pretty well, and it's not necessary to incubate it.

I have an stock of water with RNasa 20 ug/ml. I elute DNA in that water. This way, in the long run, I spend less RNasa!! I don’t incubate, it is not necessary, 5 minutes of incubation is more than enough.

aaahhh...very clever AP. I think I will try that one too!

hi
in kits, they add Rnase to a final concentration of 100µg/ml
So i have a stock at 27.5 mg/ml (don't know why the guy who kindly prepared it for the lab did a way which drives me sometimes crazy with calculation) and & add 4µl of it in 1ml of Tris 10mM pH 8.5
I prefer tris pH 8.5 because i've noticed that this is a better soltion than water for RNase treatment.