No plasmid after a maxi prep mystery - (Apr/12/2012 )

Here's a conundrum for all you molecular biology experts.

We have 7 constructs (all the same vector backbone) from the same company, we have managed to get good amounts of plasmid yield from 4 of them. The remaining 3 constructs will transform fine, but when we grow them up and then maxi prep- we get no DNA from the maxi prep.

Any suggestions, answers, help?

Are the other three constructs larger or/and contain repetitive sequences or toxic product?

Hi Trof,

In answer to your questions
The constructs should all be the same size within a few base pairs, we don't know the exact sequence- they are shRNA constructs, so although they should silence a gene, they shouldn't be toxic to cells. If it were a toxic product I would anticipate a lack of bacterial cell pellet after growing up, but we don't see that ( this part I may be wrong on). The pellets are large- there just doesn't seem to be any DNA when we maxi prep.

Make sure you keep antibiotic selection. If ampicillin, then switch to carbenicillin as the antibiotic. Losing plasmid selection gives a large pellet and no plasmid DNA.

Thanks Phage- might try that. Its a little frustrating as all the other constructs maxi prepped fine, but then didn't work in our experiment