Colony PCR is notorious for having both false positives and false negatives. Do a proper liquid culture with selection, then a plasmid prep, then the PCR. Include both a positive control and negative controls.

As my advisor said, "you can't rush quality"

Good luck!

But are there chances that I have a false negative clone? I am planning to do a plasmid prep but still wanted an easy way out. And by your answer I am guessing that colony PCR isn't reliable. Correct?

Yes you can get false negative results if you have a failed PCR. Sometimes with colony PCR you have some inhibitors that make the PCR fail. So you don't really know if you have mixed results or not.

Doing a proper plasmid prep is really quite simple and given that you have already spent a month on this, I would also recommend it. You might want to do mini preps from a few different colonies just to be sure they are all the same.

Hi Micheal, thanks for answering my question but how can I do a plasmid prep from Agrobacterium?

I don't work with Agrobacterium specifically so am not certain. But I imagine you can just try the same plasmid prep you would use for E. coli.

Generally it's not easy to extract plasmid from agrobacterium.any suggestions would be of help