No DNA in 1 of 6 plasmid preps (Qiagen - Midi) - (Sep/26/2008 )

Hi. I've done this plasmid prep twice now and I have gotten the same results. I am extracting 6 different plasmids (from 6 different cultures). 5 of them I get great DNA yields. However, the 6th one I get nothing, maybe very very little DNA. I've done everything together using the same buffers, tips, pipettors, etc.

The first 5 plasmids are around 6kb and the 6th plasmid is over 10kb. Could this have an effect on poor DNA extraction using the MIDI prep kit? How to get good DNA yield from this plasmid that is over 10kb?

Thanks!

10kb should not be a problem. maybe its a low copy plasmid while the others are high copy plasmids. Try to increase the volume of the culture.

If this is a culturing problem, I would suggest

1 - increase the volume of your culture
2 - use a bigger culturing vessel. Try aiming for a flask that is 10x larger than the volume of culture you are growing. Do not hesitate to use two flask to grow your colony.
3 - you can reduce the culturing temperature from 37 C to 28 C, this help with yields
4 - use a better medium. Use SOC, Terrific broth, or 2xYT broth instead of LB.

However I am not so certain that the fault does not lie with the column. The columns do have a maximum optimum around 10kb. Yes, it can extract bigger, but the yields are reduced.

So to get the most plasmid DNA in your culture, I would use alkaline lysis. Here you can actually see how much DNA you have.