I have a plasmid with kanamycine antibiotic resistant gene and Bar as a marker gene. I need to transfer this plasmid into AGL-1 strain of agrobacterium tumefaciens. I faced a problem when I follow the protocol steps of transformation. I did not get bacterial colony even after 2-days of a LB media plate having Kan 50ug/ml.
Protocol steps
1- Take competent cells from -80 C.
2- Add 5ul of plasmid having conc. 50ng/ul in 100 ul of AGL-1 BACTERIA.
3- Keep on ice for 30 min.
4- put in liquid nitrogen for 5 min
5- keep on heat bath for 5 min.
6- keep on ice for 5-min again.
7- add 800ul of LB without antibiotic (Kan)
8- Shake for 2-hrs at 28 C.
9- spread on LB media plate with kan 50ug/ml. Keep these plates on 28 C for 2-days.
But did not get the bacterial colony.
These are the protocol steps which I followed. Anyone can guide me where I am doing mistake?
Were the bacteria thawed on ice? What temperature did you use for the heat shock step? Have you checked the viability of your cells using a plate without antibiotic? Have you checked if your plasmid backbone is meant for agrobacterium?
Yes bacteria were thawed on ice and temperature for heat shock was 37 C. But I did not check the viability of competent cells because I bought from company so I think these will be good.
Hello, remove kanamycin from nutrient media and see do you have a grow of culture or not, if you see grow it mens kanamycin resistance gene transfection or expression problem.
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