How to treat total bacterial cell DNA with RNase - I need to get RNA free DNA (Jul/24/2006 )

Hello

I am isolating total cell DNA from Pseudomonas. I have applied RNAse at 37C for one hour right after the SDS treatment, after addition of sodium acetate. But i am getting a lot of RNA in my sample.
I think i should add RNase after i get the DNA pellet after phenol chloroform extractions. But how much RNase at which concentration should I add.

If i use RNAse after the phenol chloroform extractiosn, should i do these extractions again to get rid of RNAse after RNase treatment or just heating will be enough. Remember, i need this genomic DNA to make a library.

Does any body has an idea?

Any comments are wellcome.

Thanks for all of ur help.

yea u should add RNase after u get the DNA pellet after phenol chloroform extractions. according to my protocol u can add 10ul RNAse (from 10mg/ml stock) in 300 to 500 ul supernatant from phenol chloroform extration.

i think i dont need to do phenol chloroform extration again. but i am not sure, please check from others

What the amount of RNAse used?
I do in 10mM Tris pH 8 by 100µg/ml final conc of RNase for 30'.
Works fine

ok after RNAse A treatment i add 1 vol. of chloroform , gently shake by hand and then centrifuge at 12000 rpm, 15 min , r.t

then take the supernatant about 200 to 300ul to new tube and add equal vol of isopropanol to ppt the DNA. then wash the DNA twice with 70% ethanol and decant the ethanol and and TE quickly

You could always try adding your RNase (100microg/ml) for 30 min at 37 oC after proteinase K treatment (assuming you follow that method) that should get rid of all the RNase and you dont have to worry about getting rid of it later

enjoy

degraded RNase?
are you sure you are getting rna and not protein contamination?