Hi all, I have been facing problems with colony pcr for agrobacterium colonies. I did site directed mutagenesis, cloned to pcambia, sequenced and confirmed and then transformed to Agrobacterium. Then I got many colonies in selective media (Rif, Gent, Kan) but couldn't confirm which colony has the right plasmid with insert. For this, I tried colony PCR. The positive control (plasmid that I transformed) shows band but for colonies, no band. I took the colony in 10 microlitre water and then boiled for 10 mins and then added 3 microlitre from that as template. Could anyone suggest what to do? I know miniprep plasmid isolation would not be easy for Agrobacterium. Without confirming also, I tried to proceed with one colony but then could not express in Nicotiana.
There is probably too much DNA or bacterial debris in the PCR reaction. Try diluting the bacterial extract 100x and use only 1ul per reaction.
Here is what I do:
Put the colony in 100 ul of selective LB
shake for 1-2 hours at 28-30
use 2 ul for a 20 ul PCR
It's the same method I use for E. coli, you can use some of the remaining tiny culture to start your overnight liquid cultures too
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