Isolation of genomic DNA following RNA extraction with Qiazol? - (Apr/21/2015 )

Hi everyone,

I want to keep both the RNA and genomic DNA from brain samples.

I'm using for the RNA extraction Qiagen's RNeasy Lipid Tissue Mini Kit, and to isolate the DNA I followed a user developed protocol available through Qiagen's page (https://www.qiagen.com/pt/resources/faq?id=54b752f7-c429-41f5-bdcd-c27263ae0255). At the end of the DNA protocol I got very little amounts of DNA with poor 260/280 and 260/230 ratios.

I would appreciate any suggestions to this protocol or alternative methods to isolate genomic DNA from the phenol/interphase that you know that have worked before.

Thanks in advance,

Sara

I have done this on blood samples quite many times already (using Trizol or RNAzol)... my suggestions are:

1) as the manuals say, when you take the RNA you shouldn't get near the interphase, but after that, to extract the DNA you'll need to remove as much of the aqueous layer as possible (and just throw it away). Keep the white interphase though, because a lot of the DNA is there.

2) When you add ethanol to precipitate the DNA, do not vortex. Instead, do it as gently as possible. Add the ethanol on top of the interphase, and mix very gently. You want the interphase to turn into a white/translucid membrane. It must stay in one piece if at all possible. Keep mixing gently until the ethanol is completely mixed with the phenol, and some more. If you don't get the membrane-like pellet (because you had few cells to begin with), then you'll have to follow the original protocol and I would advise vortexing in this case.

3) I don't wash with 0.1M sodium citrate/10% Ethanol, I wash with 6.7xSSC/10% ethanol (for 3ml, it's 1ml 20xSSC, 3.7 ml water, and finally 0.3ml 100% ethanol; add the ethanol as last ingredient). Admittedly this started with a nonimportant sample because I'm lazy and I usually have a bottle of 20xSSC on the bench; the DNA I get is clean and in good quantity though.

4a) Now the important part to avoid phenol carryover, is that most of the phenol stays stuck to the plastic eppendorf tube. So when you wash the pellet with the citrate solution, you do *not* centrifuge at all. Instead, prepare an eppie tube with the new wash solution, and "fish" the precipitate with a 1000ul pipet tip (you just try to aspirate it, it'll clog the tip and get stuck; you don't need to aspirate it in the tip, in fact you want to keep it in one piece). Drop the pellet in the new tube. This will remove much more phenol, because phenol sticks very easily to the plastic eppendorf tube, so it's easier to just transfer the pellet to a new tube altogether. Even when I still have the pellet in the phenol/ethanol mix, I try to fish it out, put it in a new eppie and then remove all of the solution, then add the first citrate wash.

4b) if your pellet is very small, and you had to vortex etc, then you can't follow 4a; you need to centrifuge the pellet at every wash. But you can still add 50ul of the new wash solution, and then transfer to a new tube. Repeat to be sure you've taken all of the pellet. Add the rest of the wash solution to the new tube, etc.

Hope this helps!

Roberto