Hi,
We have years of experience cloning, but are encountering a baffling situation we can't figure out. This is happening following LR-recombinase cloning. We transform DH5a cells, and plate on antibiotic plates. We get the expected amount and morphology of colonies. We then pick colonies and grow in liquid LB with antibiotics. Again, cells grow fine. Then, after mini-prep, there is no plasmid. This is not 100% consistent, around 1 in 10 colonies gives completely normal and correct plasmid.
Anybody every experienced this and know what's going on?? We're stumped.
Thanks!
Plasmid transformation is randomly so some plasmids that were transformed reannealed and others probably knock out the plasmids may be due intolerance of certain concentrations of antibiotics
Plasmid transformation is randomly so some plasmids that were transformed reannealed and others probably knock out the plasmids may be due intolerance of certain concentrations of antibiotics
Dear Benjamin
can you provide more details about ..which plasmid, which antibiotics in which amount?
did you performed any pcr colony screening ti check for plamid presence before inoculation and mini kit to check where is the problem, if the colonies are really empty or you lost the plasmid during the propagation or extraction?
best regards
.Manuele
Hi Benjamin,
Doing a positive control (another plasmid that you may have and get very high transformation rate with it), and a negative control (DH5a "transformed" with the plasmid buffer or H2O) along with your transformation could help to figure out what is going on.
Then, the colony PCR screening should be done ( Agreed with Manuele) to eliminate any possibilities of losing the plasmid during the culturing and/ or plasmid extraction and purification.
Have you tried a new stock of DH5a? the one that you run might acquire a resistance mutation.
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