We tried twice to transform BL21 DE3 CODON PLUS with a pET plasmid, using 20 µl of cells and 1 µl of plasmid (around 100 ng), following all incubation steps (including incubation in water bath for 20 seconds) as described in the Agilent Technologies manual. After incubation at 37°C with shaking, we plated the cells on agar containing kanamycin (50 µg/ml) and chloramphenicol (34 µg/ml). After incubation at 37°C, no colonies grew. What could be the problem?
First are you sure that the pET vector you are using has that drug marker (I presume Kan in this case?). Some pET vectors are Amp-R.
Secondly, did you do any controls like a control plasmid into the same cells?
Third, are you sure you have plasmid DNA? Can you see it on a gel?
Assuming you did the procedure correct, the problem would be either the cells are bad or your plasmid is bad.
We previously transformed TOP10 cells with this plasmid, and they grew on plates containing kanamycin. We are sure that this plasmid has Kan-R. After a maxiprep, we ran a gel and observed a band around 4000 bp, even though our plasmid is approximately 6000 bp. Is that possible? We also performed a control transformation with BL21 , but no colonies grew again.
I would try some control plasmid with BL21 cells to see if the competent cells are the problem.
But if your plasmid is migrating at 4kb and should be 6kb, then I would be concerned. Double check that the plasmid is correct before you waste any more time on it.
By the way, if you were running uncut plasmid then you don't really know anything about its size. You need to do appropriate digests, an enzyme that cuts once to see the full size, and then a pair of enzymes that releases the insert and you should see plasmid plus insert bands.
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