Combining Trizol DNA extraction with QiaAMP micro purification

[zeus_dk]

Hi!

I'm working on a project were I extract RNA from frozen breast tumortissue, using TriZOL for microarray analysis. I'm interestet in a protocol were I can extract DNA suitable for arrayCGH from the remaining lower/interphase.
I've done some testing were I've extracted DNA using the Back Extraction Buffer described by Ambions TRI-reagent and then done a clean-up using Qiagen qiaamp micro column. I manage to get 260/280 ratios around 1.8.

I also want to add a RNase step, but how and when do I do that?
Does anybody have experience with something similar og have any ideas to improve the protocol?

Best regards
Zeus

[danfive]

You should be fine using the Back Extraction Method. Such as here --> [Only registered users see links. ]

Once your DNA is in TE or water you can perform the RNAse digestion, then take OD measurement.

[zeus_dk]

Originally Posted by danfive

You should be fine using the Back Extraction Method. Such as here -->
Once your DNA is in TE or water you can perform the RNAse digestion, then take OD measurement.

I have actually tried exactly that protocol you are linking to. But I think I have too little DNA, because I tend to loose it after the PCI step, even if I add acryl amide as carrier.

What would you think would happen if i just apply the upper aques phase from the back extraction method directly to the qiaamp micro column? Is that a crazy idea. People to it for the RNA extraction using the upper phase from the Trizol step and apply it directly to the RNeasy columns.

Best regards

Zeus

[danfive]

Your idea is perfectly fine.

Also note, if you are getting very little DNA, it's a good chance you have little contaminants and may not need the extra clean up steps like Phenol:chroloforms:IAA or qiamp column.
So I suggest you could also try using the First suspended DNA pellet after the back extraction buffer and isopropanol precipitation + EtOH wash.