After extracting DNA from very dirty water samples doing a double PCI extraction (with CTAB) - pellets were brown. Rehydrated samples in 30ul of TE buffer but they have a light brown tinge to it - I want to clean these samples up but not sure on the volumes of ethanol to use.
Was going to try the following:
Dilute sample up to 200ul (adding 170ul ddH20), add 200ul cold 100% EtOH and 0.2V 3M sodium acetate (so 20ul). Let it sit for an hour at -20oC then spin it at max speed for 15 minutes. Remove the EtOH and do a second wash with 70% EtOH using 200ul. Spin it, let it dry and elute in 30ul of TE buffer again.
Seen a lot of protocols out there, I don't want to mess my samples up (very precious samples!).
Any advice would be much appreciated!
No I didn't try a spin column I'd have to find a kit for that. I ws hoping to try without need for a kit. Would it still ve possible even with 30ul of DNA?
You can just volume up to 200-500 uL and do a phenol-chloroform extraction followed by ethanol precipitation. Just be sure to re-extract the phenol with TAE or water, and clean the aqueous phase after that once with chloroform. During ethanol precipitation add glycogen along with 3M NaOAc. I usually leave my samples at -80 overnight but only because it works out that way for my schedule, 1 -2 hours at -20 should be ok too. Good luck!
Sorry how much glycogen do you add? Wouldn't the glycogen precipitate with the DNA if I add it during ethanol precipitation?
5-10 ug of Glycogen.
It does co-precipitate but does not does not inhibit PCR or digests up to concentrations of 30 mg/ml, Ligation till 7 mg/ml.
I've tried cleaning it again from 100% ethanol to precipitate (with NaOAc) and a wash with 70% EtOH - pellets are still brown.
We do not have glycogen so I did not try that method.
Will try from the PCI stage again after adjusting DNA volume up to 200ul - hopefully that will work.
Yea nothing worked yet...I even did a PCI extraction again on the sample by bringing it up to 100ul with TE buffer and repeating the process. The pellets looked cleaner but I lost a lot of my sample and was really hard to even grab any without getting the organic phase.
Yea. When I work with small amounts I notice that it is very essential to have a carrier for your nucleic acids.
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