I am trying to clone a pGreenPuro vector (size 8kb) into stbl3 cells but eventually there is no colony on the plates. I plate them 37 ’C overnight on the LB-agar plates.
Any solution?
Presuming you are following a normal transformation procedure and you are not getting colonies, then you can quickly guess the the problem is 1) your competent cells are not comment; OR 2) your plasmid DNA prep is not good; OR 3) you picked the wrong antibiotic for selection. Just do appropriate controls and you can figure out the problem.
Michael J. Benedik
Dear Michael, thank you for your comment.
I used the right Antibiotic. Actually, as a positive control, I transformed Top10 cells+pUC19 and i had so many colonies but when I transformed Stbl3+ pUC19 i have a few colonies.
it seems stbl3 can’t grow properly.
The Stock solution of competent cells is completely new.
Then it sounds like your problem is with the Stbl3 competent cells. You could ask for a new batch of them, but are you sure you really need to use that strain?
Michael J. Benedik
Yes, the problem is that stbl3 cells can’t grow properly. For transforming the lentiviral vectors, it's highly recommended to use stbl3 cells.
Does your plasmid transform Top10 as well as pUC19 does? it may be that you have both problems, low plasmid and not highly efficient cells.
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