I am working on a 11kb plasmid and trying to get a qiagen prep of it. I
have tried twice once using high-copy plasmid protocol with qiatip-110
and once using low-copy plasmid protocol with qiatip-100. Both times I
did not get ANY DNA from the preps. I know that my techique is right
because I ran other plasmid preps parallel to this one and got tons of
DNA for it. Hence I think the problems that I am encountering are
specific to this 11kb plasmid.
Let me tell you more about what I think.
1. This plasmid grew well in LB AMP (100ug). This was the culture I
used to prep qiagen DNA and got nothing. I streaked a plate from this
culture and suprisingly nothing grew. I always thought that liquid
culture is more stringent than agar plate and it is very surprising to
see that the culture that grew in liquid AMP did not grow on the plate.
May be that is the reason I did not get DNA but how come the cells grew
in liquid culture
2. I also don't think that this plasmid has gone really low copy
because I have a miniprep from 1.5ml of the original culture (the
culture that I talked about above was scaled up from this original
culture). I digested 3ul of this miniprep and I could see good amounts
of DNA on the gel. So my guess is that somewhere in the middle the
plasmid is lost.
I have plans of retransforming the miniprep I have, testing that to see
if it has the plasmid by digesting and then use that retransformed
cells to scale up my culture for qiagen.
It would be great if I can get more suggestions to get this 11kb