No DNA after midiprep - (Feb/17/2012 )

Hi all,

So, this is driving me mad and I hope that somebody can help me.
Basically, I have NO DNA after my midiprep. This is what happens: I checked several minipreps, which contained the construct I desired (good yield, perfect restriction). I use a bit of the miniprep culture (stored o/n at 4 degrees) to inoculate a midiprep (100 ml), do a midiprep with the Machery-Nagel kit and NO pellet. The midiprep culture was very cloudy, so plenty of bacteria. Also, in parallel I midiprepped another construct (although different backbone), which gave a beautiful pellet and good quality DNA, so I can midiprep and the kit is OK. I repeated it three times now, still no luck.

Is it possible that with this particular construct is kicked out by the bacteria (but then I don't understand why my minipreps had no troubles)? I tried with adding more Amp (freshly thawed), increasing the volume to 400 mls.. The only thing I can think of now is to retransform the miniprep DNA, but I am afraid this will eventually give the same problems in the end. Or growing for a shorter period?

Thanks!

Take 4ml of your midiprep and isolate with miniprep kit, and see if you get any DNA.

how long do you shake the cultures? Longer shaking periods can at times lead to similar results.
Try shorter times.