Loss of insert? - (Oct/28/2011 )

Hi all

I have been experiencing some cloning problems, if someone could give some suggestions. I am trying to clone two PCR products individually (from different genes and both around 3kb) to a 7kb plasmid. I used SacI site for one, and XbaI for the other one. After restriction digest, CIP treatment of vector, and ligation, I got 2 to 3 on my vector only plates, and got 20 to 50 colonies on insert+vector plates. Things are going well at this step for both cloning.

When I mini prep them, and cut plasmids with SacI or XbaI, respectively. I can't get insert back, instead the plasmid got linerized. It seems like that plasmid doesn't contain insert, or one sites got destroyed during cloning?

I have used this plasmid to clone other big fragment at either SacI or XbaI sites, and all worked fine.

Does anyone has any idea why that is?

Thanks

CD

SacI can be a tricky enzyme ...the most frequent problem encountered is salt inhibition ...so if your miniprep's contain residual salt than SacI will be inhibited.
For details see the NEB page!
Additionally, SacI cuts supercoiled DNA not that well so you need more enzyme ...see here!

Is the plasmid size correct when it is linearized? (size=vector+insert?)

Overall i would go for a specific restriction enzyme that only cuts once in your insert ...and identify positiv clones that way!

Regards,
p