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question on DNA material?

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question on DNA material?

Postby bluerose » Wed Aug 08, 2007 4:17 pm

I am doing an E. coli plasmid prep on some transformed colonies isolated from agar media and grown up in a liquid culture. I use an alkaline lysis protocol to get the DNA out of the cells, wash with EtOH, then finally resuspend in TE. But before I do resuspend in TE, I see a clean while pellet at the bottom of my eppendorf tube that I had just centrifuged. When I do finally resuspend this pellet in TE, some of the pellet easily resuspends in TE and I can't see it anymore. The rest of the pellet doesn't dissolve so easily and looks like a thick white material, similar to what looks like a small piece of Ivory soap (not saying it is soap, but that's what it looks like!). I can break this thicker material up also and resuspend it in the TE, but I am worried this is not DNA material, but junk.

Does anyone know if this is DNA?? If it's leftover junk, how should I get it out to have only DNA resuspended in TE? Thanks.
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Postby blcr11 » Wed Aug 08, 2007 9:08 pm

Just alkaline lysis? With phenol/chloroform/isoamyl extractions, I presume. No membrane-binding step?

Most likely you took some of the interface when you removed your aqueous phase from the extraction(s). The white, waxy-looking pellet that doesn't dissolve in TE is probably residual protein. You can just spin it down and transfer the supernatant to a fresh tube. But check the prep on a gel to make sure that the plasmid is intact and that it isn't contaminated with RNA or chromosomal DNA. You should have added RNase to the lysis buffer so RNA contamination should not be the problem--and RNA should be soluble anyway, so that isn't what the white stuff is. You also want to make sure that you don't transfer any of the while, flocculent material to the lysate after you spin it out following lysis and neutralization.

If you are doing alkaline lysis without membrane binding, you should be doing 1 (or 2) phenol/chloroform extractions followed by 2 or 3 choroform/isoamly alcohol extractions before you do the ethanol precipitation step.
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