really dumb question that's making me batty - (Nov/07/2005 )
Hey y'all, I'm stuck on something sillly and I'm hoping you can help???
I'm doing a phenol/chloroform extraction of some chromosomal DNA from S aureus
I haven't done a p/c extraction in a looonnnngggg time, like probably 7-8 years, and it is not something I ever did routinely. I am following Maniatis' methods because I dont' really have time to order any silly kits and wait for them to arrive, I need the DNA yesterday.
I did the whole equilibration thing with the phenol, and followed all the instructions, except I did not add the hydroxyquinoline because it didn't look important for what I am doing.
Well, I extracted that damn lysate about 15 times and I can't get rid of the protein interface. I have gobs of DNA in the aqueous layer (as evidenced by the clear snotty stuff), but I can't pull them off without keeping most of the protein, even after extended centrifugation. I have about 500ul lysate, I am extraction with an equal volume of p/c/iaa (25:24:1) as specified in the protocol. I have even tried spooling the aqueous layer, but threads of protein precipitate come out with the DNA.
Would increasing the volume of both layers help? Another thing to note, I do not want to shear my DNA and so I am not vortexing to emulsify, I am inverting about 10X to 15X prior to spinning. Is there a better way to do this?
Does anyone know what I am doing wrong? it shouldnt' be the hydroxyquinoline, that is more relevant to RNA purification, if I am understanding it's function correctly?
any help you could give me would be greatly appreciated! I feel like a moron, I used to know how to do this.....
To me it seems that you are not just pulling out the DNA but also the cell wall polysaccharides, which are often complexed with proteins. I would suggest using phenol:chloroform: isoamyl alcohol and perhaps starting the extraction in a PVP buffer rather than the usual lysis buffer.
Works for plants anyway, can't say I have had that problem with bacteria before, myself.
I'd suggest that you take the supernatent to a new tube, and then re-extract a second time with phenol, or, perhaps better, with phenol-chloroform. Multiple extractions work better than trying to optimize a single one. You can do this until a clean interface occurs. Finally, do at least one chloroform only extract to get rid of the phenol, prior to the ethanol prepcipitation.
I would try increasing the volume of both fractions. Also use a wide bore tip (cut the end off if necessary) to reduce the pull. I have had a similar problem where it was extremely difficult to remove the aqueous phase due to the viscosity but I was working with much larger volumes (3-5ml) so I just left some behind, I was also using a graduated pipette and being extremely gentle applying as little vacuum as possible.
I have scaled up the volume and taken part of the DNA; there is still a tiny bit of white stringy stuff but I found a reference suggesting perhaps it's due to the high DNA concentration (although I am skeptical...I'll be running the gel soon and find out if the prep is any good)
I extracted with p/c/iaa (phenol/chloroform/isoamyl alcohol at 25:24:1) over a dozen times with each sample, with a transfer to fresh tube each time. I added a couple c/iaa at 24:1 extractions at the end to remove the phenol. This did not give me separation, it was what I had tried before I posted this yesterday.
Yesterday afternoon I scaled up the volume. Hopefully it worked and is not loaded with a bunch of protein; I'll know soon. I also used a wide-bore pipet tip and worked carefully; the last thing I want is to shear it after all this PIA.
for the plant gal, what is PVP buffer?
thanks for all the replies!
PVP is polyvinylpyrrolidone, comes in a range of molecular weights, most people use PVP-40 (Mr=40,000) or PVP100 (Mr 100,000).
p.s. Guy not Gal
sorry, bob! I was reading through in a hurry and saw the 'amanda' before I replied.....
Well, the gel was good and bad...out of paranoia about the proteins in the interface, my yield with two of the three preps sucked, but the third turned out just fine
I decided to quit monkeying around with something that should be so easy....I ordered up some protein extraction solution stuff from promega (part of their Wizard kit) that replaces the P/C step, then I don't have to worry about separating any layers and all the nasty waste
but, thank you all for your replies and help!