Endofree DNA precipitation accident-- can I salvage my dna? - (Jun/13/2008 )

Hi all,

I'm very new to the whole lab experience, and I feel like I'm learning everything by trial and lots of errors. Anywho, I am using the Qiagen EndoFree Maxiprep kit to isolate DNA for a retroviral transfection. We've already done the transfection once, with unbelievably low transfection effeciency. That is most likely due to errors in my technique, but it was suggested that we use endotoxin-free purified plasmids.

Thus, today's maxiprep; however, when I got to the DNA precipitation I added 10.5 ml EndoFree 70% ethanol rather than isopropanol. I talked to a tech at Qiagen, and he suggested I just continue from that step (ethanol wash) on and to expect very low yield. He also said it might help to centrifuge longer (the protocol says 30 min w/iso, 10-15 min for ethanol wash) so I ended up centrifuging the eluted DNA in 70% ethanol for 1 1/2 hours in hopes of getting a pellet. There's barely anything in any of the tubes (I was doing 5 preps). The best thing we could come up with to do was top the centrifuge tubes off w/100% ethanol and spin them down after a weekend at -20.

I've saved my cultures and will probably end up redoing the prep Tuesday (so expensive ), but I was wondering if anyone else had any advice.

Sorry for such a lengthy post,

Newbie

Sorry to hear about your mistake. I call this learning by "doh" (Homer Simpson style). This is one of your most valuable learning tools as once you make the mistake, chances are good that you won't make it again. Anyway, I would treat your samples as if you were doing an EtOH precipitation. Add sodium acetate pH5.2 (final concentration of 0.3M) or sodium chloride (final concentration 0.2M) so that the DNA will precipitate. Leave it in the freezer over the weekend and then centrifuge. Wash the pellet with 70%EtOH and then resuspend in desired volume of water or TE. At least this way you may be able to make a small but concentrated DNA prep. However, I've never done this and you may be best just starting over with the cultures. DNA preps aren't that hard to redo. It's when you mess up a huge time course (around the last time points at 3:00 in the morning) that makes you want to go play in traffic.