I performed electroporation to introduce a plasmid into Agrobacterium and then plated the transformed bacteria onto media containing antibiotics to select for the presence of the plasmid, but subsequent PCR reactions for the target gene are negative
Some good controls will help you with troubleshooting. I've had a plasmid end up being toxic in my bacterial cells so I got a few colonies but all were empty. How I figured that out was by using a different plasmid with the same selection (and of a similar size). That second plasmid transformed fine while my plasmid of interest did not. For me, the key information was the low number of colonies that grew from my plasmid and the high number of colonies that grew from the control plasmid.
It is also challenging to do colony PCR on Agro. You'll need some really good controls that also use the plasmid as a template. Often plasmids have set primer sites (like M13F and R). Those should help.
I like to take a bit of the colony, put it in 100 ul of media with selection, vortex, outgrow for 1-2 hours at 28, then use 2 ul in a 20 ul PCR reaction.
Amy Klocko, I have no problem with Ag PCR, The problem is that I have colonies which are able to grow on the media with antibiotics but with PCR they are negative, This never happens to me before.
Hmm, are they empty vector plasmids or are the plasmids entirely absent? The bacteria should not be able to grow if the vector is absent . . . any chance your antibiotics are getting older? They do expire.
If you have a set of primers that can detect the plasmid in general that might help.
The plasmid is entirely absent, I did plasmid extraction and then PCR compared to the PCR of the purified plasmid (+ve control)
Dear Mohamed Samir Youssef did you try to pate your Agro without transformation or a mock transformation to see if there is some contamination that is not Agro or an Agro resistant to your antibiotic of choice or that antibiotics are ok? After purifying plasmid from Agro, do you run it on gel before doing PCR? normally the concentration is very low, but you could still try to see if you got any plasmid at all or nothing.
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