PCR of MIE-14v just to make sure that we can’t get a product from this sample, despite NanoDrop readings suggesting that there’s no DNA. Used both LABY and Euk primer sets. PCR set up is here. Anneal temp 50C.
Lane 1 – 100bp Ladder
Lane 2 – Euk
Lane 3 – Euk H2O
Lane 4 – Euk H2O
Lane 5 – Euk H2O
Lane 6 – LABY
Lane 7 – LABY H2O
Lane 8 – LABY H2O
Lane 9 – LABY H2O
Lane 10 – 100bp Ladder
Results: Nothing, as expected. Need to devise a new method of isolating gDNA from these “problem” isolates.
Sample was pelleted by spinning in a microcentrifuge @ max speed, 4C for 30mins. Supe was removed and sample CAREFULLY washed with 1mL 70% EtOH. Sample was spun in a microcentrifuge @ max speed, 4C for 10mins. Supe was removed, sample brought up in 10uL of TE and spec’d.
Results: Nothing. Absolutely no DNA in this sample at all. It’s odd that the Qiagen Kit procedure (even without the lysozyme treatment) has worked on all the other Dungan isolates, but not this one. I wonder if the EtOH storage is having an effect on the cells; lysing them for some reason? Maybe the cells should be sent to us in culture medium instead?
Added 1/10 volume of 3M NaOAc (pH = 5.2) to sample (10uL) and then 2 volumes of ice cold 100% EtOH to samples (220uL). Mixed thoroughly and incubated O/N @ -20c.
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…