Midi-prep problem - (Oct/22/2007 )
I don't know whether you have ever had this kind of problem in midiprep... When I tried to grow the bacteria(DH5a) in liquid LB, at first I found the culture grew extremely slow(the media looked transparent after 8hrs). So I let the culture grow for 20hrs... Then I harvest the 25mL culture for Midiprep. When I did the isoproponal precipitation, no precipitates came out... It is really weird that I did four different plasmids and three of them didn't give me any precipitate of DNA but only one did. I used 50ug/mL Amp as antibiotics. It really seems weird to me because the bacteria grow normally on Amp plates... Do you have any idea of this type of problem? Thanks in advance.
Hi,
I think you should use more bacteria for midiprep, for instance 50ml culture. And if you know that you would get low yield of plasmid, you could combine 2 or 3 cultures at the end when you resuspend DNA in water or TE.
Have you quantitated your yield? The precipitate may not be very visible sometimes. i usually use 100ug/ul Amp. I suggest doing a serial determination that your transformed bacteria grow well in to determine the optimal amount of Amp to add to ensure that your bacteria grow well.
How sure are you that there's no precipitate? Midi-prep DNA is transparent and sometimes easy to loose for this reason. as is been asked before, have you check the yield? What's the difference between the 3 without precipitate, and the one that had. I'd resuspend all in 100ul anyway, and OD or run agarose gel to confirm (less tedious than start from scratch). Also I usually grow 100ml cultures for midi-prep overnight.
I'd say that 20 hours is way too long to grow a culture. After 20 hours of exponential growth in a small volume, most of the bacterial cells will have died and lysed. For midipreps to work, you need intact cells that will release plasmid only after alkaline lysis.
After 20 hours your cultures may look thick and goopy, but this is due to lysed cell debris. When you initially spin down the culture, most of your plasmid will be in the supernatant and lost.
For midis I grow a 50 ml culture for 14-16 hours max, and usually harvest before the culture becomes too turbid. My yields and purity are always great.
Ginger
Thank you all. I'll try again:)