Hello Hwee-Leng,

colony PCR is sometimes quite tricky. The reason for this is the sometimes poor quality of the colony material (presence of contaminants, genomic DNA, etc.). If you get a negative result from a colony PCR it is often possible that your clone is still positive. It is very important that the colonies you use are freshly grown (that is overnight growth). If possible, always include a positive (e. g. purified plasmid DNA) and a negative control (containg all components except template DNA) in your experimental set-up. A common error is that people use too much colony material. Less is more when it comes to colony PCR.

Good luck and best regards, Christian

If PCR positive control or clinical sample known to contain adequate number of PCR specific target molecules for visualization after gel electrophoresis/real time PCR, generates no amplifier , then the test result is regarded  as false negative. False negativity in PCR assays may be facilitated by number of mechanisms normally problems associated with target molecule, the PCR mix, the primer design, the thermocycling protocol or the sensitivity of detection system used. Reagent artifacts  and colony inserts also give false results.

Noted. Many thanks. :D 

If it is important, then you could use a second set of primers that amplify a part of the plasmid backbone. Failure to see that band would tell you that the the PCR failed on that sample and not that you didn't have an insert.