DNA purification - Touble with isolating PCGP plasmid (Dec/18/2008 )

Hi,

I've been having trouble making pcgp plasmid (gag-pol-env in pCI vector). I transoformed TOP10 cells (invitorgen), then used qiagen maxiperp. I got nothing. In qiagen mini-prep I see the plasmid. Did anyone have the same problem and what's you solution? I have a lot of experience with maxis etc. I was thinking it may be the TOP10 cells?

Thanks!!

Hmm.... assuming that the maxiprep columns are not at fault, I would guess it is the plasmid which is causing the problem. The cell don't like the plasmid.

In this kind of situation, I would
-innoculate the maxiprep with a fresh culture of your plasmid containing cells,
-decrease the incubation temperature from 37 C to 30 C.
-use a richer medium than LB, eg TB, SOC, 2xYT
-if possible use a bevelled flask
-if possible use a flask that is 10x the volume of the culture volume. I would use additional flask if needed to obtain the desired culture volume

Personally I would switch to the traditional alkaline lysis method. This method allows you to monitor what is going on. How much plasmid do you have.

In the best one I got 7.5 microgram total (!). I always inoculate from a fresh culture, and not glycerol stocks. What is a bevelled flask (is that the flask that enables the culture to be airated?). I get growth but not a pellet after isopropanol. The kit/isopropanol is fine, I just made other maxis which are good.
I think maybe to increase lysis/neutrelizing buffers to pellet ratio? I do not have experience with TOP10, are these good for qiagen maxipreps?


Thanks!!!

Check the replication origin of the vector. Low copy number vectors will give very low yields on a maxiprep. You may want to try chloramphenicol amplification if you have a low copy number vector.