Agrobacteria

[amandazzle]

I recently did an electroporation on agrobacteria. The construct had two inserts that I screened for using colony PCR. Unfortunately, the PCR indicated that while the original plasmid had both inserts, the agro contained only one. I repeated the entire process and the results were the same. What is going on here? Has this happened to anyone else? Could this have anything to do with the strain (LBA4404) of agro used? Could this be a "false" negative in the PCR?

[canalon]

Well I don,t know your PCR setup, but if I suppose that your positive control was correct (Hence the problem is not in the PCR mix) and that you amplified the same template in both PCR (and then you do not have an extraction problem) we must assume that there is something wrong with your template. But I have no Idea what it can be.

[amandazzle]

Both the positive and negative controls were fine. The PCR was not a multiplex, so I just ran each set of primers individually on the colonies.

I work in Brassica transformation and cell biology, so I don't design the constructs, I only verify them either with a digest or with PCR before transformation. I have considered carrying out the transformation on the plants to see if the gene will be present, but wanted to find out if there was any other troubleshooting I could do before declaring defeat or wasting my time.

Is there any chance that only one insert is present? I can't think of how that would happen. I am leaning towards a false negative.

[samuelmain]

Hi,
I will tell you my experience : I used to test my colonies by a single PCR, but i had too much troubles with false positives. Now, what i do is 1) colony test by PCR 2)mini-cultures of 3 to 6 positive clones 3)miniprep and restriction analysis (more time consuming, but much more informative) and 4)transformation with those which are still positive. Now, no problems. Try this procedure, you might be surprised by the results of the restriction screen.
And tell me your results (good or bad)!
Good luck and be brave,
sam

ps : please excuse the poor quality of my english. Tell me if there is anything you don't understand.