4 replies to this topic

[LostintheLab]

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?

[Trof]

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

[LostintheLab]

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.

[phage434]

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.

[LostintheLab]

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