Hi, I have grown BL21 cells with the pET28a that carries my insert sequence. It is growing on the selection plate but I can't observe any insert sequence bands when I do colony PCR. Is there any possibility for the BL21 bacterial cells to expel the plasmid out after the expression?
Dear Tasshitra Subramaniam
how long is your insert? Are you sure that is not the PCR the problem? Did you performed a positive control with the pure plasmid?
did you screened several colonies?
if you are growing the cells under selection of the appropriate antibiotic the probability that all the cells lose the plasmid or the plasmid is recombinated and lost your gene is very low.
Since BL21 is not a recA strain is it possible that this may happen after several cicles of growth, but after just one is very strange. I think that there are higher probability that something in your PCR screeinig failed.
best
Manuele
we analyse/sequence /characterize our plasmids from a large clean prep that have been produced in DH5a (or similar strains made for DNA production), then transfert it in BL21DE3 for expression: we never check the plasmid in BL21 (they are poor plasmid producer) exept for antibiotic resistance...never stock the plasmid in BL21 always start pre-culture and induction from a fresh clone from a fresh BL21 transfo on a plate: of course there is case where the expression of the gene is toxic and the plasmid is progressively lost in BL21 during large culture but generally there is enough cells to produce your protein of interrest then you can lower the temperature of cell culture, use more tighly controled expression...
Colony PCR is notoriously unreliable. It's easy to get false positives due to residual insert on the media surface & also easy to get false negatives due to incomplete lysis/too many cells. Pick a few colonies & streak onto a new plate of selective media. Then, do an overnight liquid culture under selection & a plasmid prep. You can't rush quality. It's possible for colonies to "drop" a plasmid if the antibiotic degrades or if you get satellites.
Good luck!
Thank you for all the suggestions. I did colony PCR again with the pure plasmid of ~10ng as the positive control. But this time, I changed the PCR setting (increased the duration of initial denaturation) and reduced the volume of the plasmid, which gave me positive results.
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