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Agrobacterium LBA4404 transformation - (Nov/22/2012 )

Good day everyone

Im currently transformed pGSA1131(binary vector) into agrobacterium competent cells. Since agrobacterium resistance to Rifampicin and pGSA1131 resistance to Chlorophenicol thus both antibiotic was used for colonies selective after transformation.There was a lot of colonies growth on petri dish with selective antibiotics (15ug/ml for rifampicin and 30ug/ml for chlorophenicol), BUt none of it given a positive result through PCR colony.

Does anyone faced similar problem as mine? Please kindly reply me..Im realy appreciate for your help. :)

Thanks in advance.


If you meant that you tried colony PCR, then this is not reliable, especially as a negative control. Many things can make a PCR reaction not work. Instead, do a miniprep, and look for correct bands. The usual way colony PCR fails is that people use far too many cells. You could try suspending a colony in 100 ul of water and using 1 ul as template in a PCR reaction. The initial 95 to 98 (before you start cycling) heat period should be extended to 3 min or so.