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DNA extraction with phenol-chloroform - DNA stuck in phenol layer?

microbiome DNA extraction phenol-chloroform phenol chloroform

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#1 harora

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Posted 24 January 2017 - 08:03 AM

I recently performed a DNA extraction for a microbiome project from cotton rectal swabs and urine pellets using phenol-chloroform. Briefly:

1. Swabs were incubated with a mutanolysin/lysozyme enzyme cocktail for 1h @ 37C

2. Added glass beads, phenol:chloroform:isoamyl alcohol (25:24:1) and 20% SDS, then agitated (TissueLyzer/bead beater) and centrifuged @ 14k rpm x5 min. 

3. Aqueous layer transferred to a clean tube, and added 3M sodium acetate and ice-cold isopropranol at -20C overnight.

The next day, no pellet! I pulled off the supernatant carefully (assuming there might be a pellet I couldn't see) and re-constituted in water, ran a PCR and a 1% agarose gel but no DNA bands! I ran the sample on the UV-Vis (Nanodrop) and got nothing (no peaks, no DNA). 

I also tried UV-Vis on the supernatant and got what looks like a phenol peak at 270 and also a second peak at 260, which I think is DNA (see attached). 

My interpretation is that the aqueous supernatant (with NAOAc and isopropanol) still has both phenol and DNA in it, the former being a technical error I suspect. I've tried adding more NAOAc or isopropanol or even repeating the phenol-chlor extraction with aliquots of this supernatant, but to no avail. 

Does anyone have suggestions on how to pull the DNA out of this solution?

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#2 mdfenko

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Posted 25 January 2017 - 04:25 AM

you don't mention how hard you centrifuge after the overnight precipitation. maybe harder and/or longer will pellet the dna. also, sometimes the dna pellet will stick to the side of the tube as a sheet (not very visible). you may have to flush the side of the tube to recover dna.

 

you may be able to extract the dna from the aqueous layer with glass milk or something similar (spin columns).


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