plasmid isolation - (Aug/27/2001 )

Dear Colleagues:

My work is stock on a trouble for plasmid isolation. Could any one give me some suggestion or comment? I cloned one DNA into vector, after transformation, the negative control was clear, no bacteria grew.The positive control grew very well, and my experimental group grew about 40 clonies in one plate. But when I did miniprep of plasmid by QIAGEN kit, I could not get any plasmid. What happened? Thank you in advanced!

Duan

I also meet this problem,but when I repeat it from the transformation,it can work again.So I suggest you can try it again,maybe you will get it!

I think both you and "apoptosis" may have "thrown the baby out with the bath water" as we say.In other words, you may have lost your plasmid with your supernatant from one of your wash/precipitation/centrifuge steps.Try to be more careful when removing the supernatant from your pellet. Always orientate your centrifuge tube the same way, for example, with the hinge outwards, so you know where the DNA pelletshould be. And always reserve and label your discarded supernatant so you can always go back to it if you do make a mistake.

sometimes the plasmid gets integrated into the bacterial genomeanother possiblity is that u have got revertants,start afresh!