Colonies appeared after transformation of pHT01 plasmid into Bacillus subtilis, and band was confirmed by performing colony PCR. However, if the colony is picked and grown (in LB+antibiotics) and then colony PCR is performed again, the band does not appear. I think it's probably missing the plasmid, can anyone explain this phenomenon?
Was there no-template control in the first colony PCR? First positive results could be a consequence of contamination.
Was the plasmid purified before the second colony PCR? If so, it is beneficial to check its quality by gel-elecrophoresis.
Colony PCRs can give false positive results. When you add the ligation mixture to the competent cells for transformation, there will be many uninserted GOI DNA in the mix. These fragments of your GOI get plated along with your competent cells and it is possible to pick them along with your suspected clone for colony PCR. This gives you a false positive; especially if you have added a higher molar equivalence of insert to the vector. You will have to redo the colony PCR.
If the problem persists, skip the colony PCR step, isolate the plasmids manually and verify by regular PCR with the plasmid or double digestion.
I agree with @ Praveesh Valissery, and do not recommend colony PCR.
Your desire to screen the positive transformants could force you to do regular analysis through the cultivation and isolation of DNA. For example, microalgal chloroplast transformants should be tested through PCR for GOI integration, Southern blot for the single copy, and Western blot for the stable expression.
Many thanks for the kind advises.
The plasmid was constructed in E. coli and preparated plasmid was used in the transformation to B. subtilis. The colony PCR was conducted with negative control (wild-type B. subtilis) and positive control (plasmid). In addition, the other gene in the chromosome of B. subtilis was also used as a positive control of the colony PCR. Therefore, I think the colony PCR was done correctly and the plasmid was segregated from B. subtilis. Is there any other advise?
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