How to avoid RNA contamination in DNA samples:? - (Aug/08/2013 )

Hi!

I would like to know if there is a way to avoid RNA contamination during homemade Midiprep or remove from an eluted DNA sample. 

I use RNAse and it seems to work well, but I´m still seeing contamination when I run an agarose gel.

My precipitation protocol is the following:

- I usually have 6 ml of lysate and I transfer it  into 6 microtubes (1 ml each);

- Add 500ul of isopropanol (room temperature); mix well;

- Spin 15', 16000 rpm, 4*;

- Remove the flow-through;

- Add a total of 1ml of cooled ethanol in the 6 tubes and unite the pellets;

- Spin 10', 16000, 4*;

- Remove the ethanol remaining;

- Spin 30";

- Add 500ul of TE 1:0,1 and incube for 1 hour at 37*;

- Add 8ul of linear acrylamide and 2,5 ul of 20mg/ml RNAse;

- Incube 1 hour, 37*;

- Add 200ul of MgCl2 50mM;

- Add 500ul of PEG 8000/ 30% (final 12,5%)

- Spin 15', 16000, 4*;

- Wash twice with 1 ml of ethanol

- Spin 10', 16000, 4*;

- Remove the ethanol remaining and spin 30";

- Dry the tubes at room temperature for 5';

- Add 500 ul of TE 1:0,1 and incube at 37*C for 1 hour.

Any suggestions?

Thanks!!

The RNase step in these sorts of protocols is usually done during the lysis, immediately after the protein and genomic DNA has been precipitated and spun out.

Are you sure this isn't genomic DNA that you are seeing?