gDNA Isolation – Dungan isolate MIE-14v

Cells stored in EtOH were pelleted 5000g, 10mins, 25C. A brownish smear was present along the inside of the tube after spinning; not really a pellet per se. Supe was removed and cells were washed twice with 1X PBS. The smear was reduced to a pellet after the first wash in PBS. The second wash resulted in a slightly smaller pellet, but a pellet was present nonetheless before proceeding. Cells were subjected to an enzymatic lysis in 180uL of a TE/Triton X-100/lysozyme mixture as described in the Qiagen DNeasy Kit for Gram-Positive Bacteria (and for cells having substantial cell walls). Cells were incubated in this mixture for 30mins @ 37C. 25uL of proteinase K and 200uL of Buffer AL were then added and the mixture was incubated @ 70C for 30mins. Protocol then followed the “normal” steps for isolation of gDNA. Sample was eluted in 100uL of Buffer AE and then spec’d.

Results: Well, it doesn’t look like there is any DNA at all… Will try precipitating the sample and bringing up the DNA (if there’s any) in a small volume. It should be noted that I spec’d these samples using the “RNA” setting, so the constant for concentration calcs is wrong (40), but doing quick math using the DNA constant (50) shows that there’s still almost nothing there…

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