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Smeary restriction digest of genomic DNA

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Smeary restriction digest of genomic DNA

Postby icequeen11 » Tue May 22, 2007 7:43 pm

Hey all, same prob as a recent post, but working with genomic preps (from fish muscle tisue) instead of plasmids. I ran a digest with a single enzyme (Sac I) on 8 different samples, and did an incubation with the specified buffer as a control for each sample. The same tubes of BSA and buffer were used in all digests and controls, and all samples were subject to the same physical handling (temperature, flicking, spinning, etc).

5 of the 8 digests and their respective controls came out smeary, complete with low molecular weight bands indicative of histonal DNA. I'm pretty sure it's more due to nuclease contamination than mechanical shearing, though there may some shear as well. The original preps were also run on the gel, and look fine for all samples. Because the same reagents were used for all samples, the problem seems sample-specific.

On my preps, I did proteinase K treatments > phenol/chloroform extractions > extended dialysis (MWCO 3500) > RNAse A treatment (DNAse free, as per Maniatis) > proteinase K treatment > phenol/chloroform extractions > extended dialysis.

My question: when doing phenol/chloroform extractions, if you pipette right down to the interface when removing your genomic fraction, can you take up nucleases that subsequently refold?
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Postby blcr11 » Wed May 23, 2007 3:18 pm

There will always be some streaking when you digest genomic DNA, but I gather there is too much very small MW stuff and not very much higher MW material. It does sound like a sample-specific problem and the simplest explanation of that is nuclease contamination--as I think you've already guessed. I can't say whether the phenol/aqueous phase interface is the source of the contamination or not. I think it is prudent to err on the side of leaving more of the interface behind--especially if there is a heavy ppt--in the early extractions. Once you've done the initial extraction you are less likely to have to worry about protein carry-through. But if you still have nuclease contamination after Proteinase K treatement?? Hmm. Sure your Prot K is in good shape? It's possible that your plasticware is a problem, but if say your pipet tips are crummy, I'd expect a kind of random nuclease activity--sometimes it's in the buffer control but not the SacI digest, sometimes it's in both, and sometimes it's in neither--that sort of thing, and I assume that's not what you saw. Still, I would try and use tips and tubes that are certified to be nuclease-free. Don't know if that helps in any way, but that's my 2 cents.
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