Phase separation during DNA extraction - (Apr/19/2012 )
I have been extracting whole genome DNA from hard tissue (brain samples) using a chloroform and nucleon resin protocol. However, after adding the neucleon resin and centrifuging for 5mins at 3200rpm the phase separation is not very clear. I think this problem is resulting in very low concentration of DNA at the end.
For example after adding nucleon resin and centrifuging, I expect to have the following phases in descending order: DNA-containing upper phase, Solid stratum of nucleon resin with bound proteins, and Protein-containing organic phase.
Instead I seem to have (time and time again) some sort of cloudy white stuff (i believe this is the protein) between the solid stratum and the aqueous phase. This cloudy bit is big, making the aqueous phase I need to collect very small. As a result my DNA samples have been giving me very little concentration.
Could you tell me what I'm doing wrong?
P.S: Here's the protocol I use
- 2ml lysis buffer (for a 200-300mg tissue)
- 200ul SDS 10%
- 20ul RNase A <50ng/ul>
- 5ul Proteinase K <20ug/ul>
- 600ul Sodium Perchlorate 5M
- 3ml Chloroform (COLD)
- 600ul Nucleon Resin (**problem is usually encountered after centrifuging/ while tring to collect the aqueous phase with the DNA)
- 2 volume of EtOH 100%
- Leave samples at -20°C overnight
- Wash with EtOH 70%
- Dry pellet and dissolve in T10E1
Am I doing something wrong? My DNA concentration has been really low even though I get an ok size pellet after leaving it at -20°C to precipitate the DNA. Any idea what I should do better?
Many thanks in advance
Brain tissue has a lot of fats. You may want to do a preliminary extraction with chloroform to remove some of the fat prior to starting your DNA extraction.
Yeah, a defatting step first would probably be most appropriate.
I'd either go with a chloroform extraction (equal volumes of chloroform and MBG water and retaining the upper phase after centrifugation) or a mixed xylene extraction (as with chloroform but retaining the lower phase after centrifugation). Xylene would be a good choice if you've wax embedded your brain tissue.
Also, on a practical note, make sure you've chopped up the brain tissue as finely as possible. Often in defatting the tissue rolls up into a little (brain-like, aptly enough) ball at the bottom of the reaction tube protecting most of the fats from the action of the organic solvent.
Also, chopping up finely will help during the phenol extraction too.
Thank you both for your reply. I was under the impression that the detergent would take of that but it seems like I was wrong. And it makes sense that I should remove the fat according to your suggestions.
So at what stage during my protocol do I do the fat removal procedure? Does it make sense if i follow my protocol (shown above) upto the point where I add Chloroform and centrifuge to get the fat emulsion, remove the emulsion and continue with the protocol (i.e. just add the Nucleon Resin and centrifuge to do the phase separation)?
Or are you suggesting I do the fat removal step before I even grind the tissue?
Thanks for your help again.
No, certainly not before you grind the tissue.
You have a choice - either right at the very start (grind the tissue, conduct chloroform extraction to defat the tissue and then, ensuring you've no chloroform in your sample, conduct as if you've just ground the tissue) or conduct it where you add the cold chloroform in the protocol (it's not clear in the condensed method you've printed what you do with the chlorinated sample but I'd think that here is a decent spot to conduct the defat step).
Also, dunno why I said phenol. Remembering the good old days I suppose.
Thank you very much indeed. I will do this step. Also I realised that my last sentence in the previous message was kind of dumb after I sent it :-).
P.S: After adding the chloroform as stated in the above protocol I usually vortex it well and add the Nucleon Resin and centrifuge it (5mins @ 3200rpm) to recuperate the aqueous phase.