Hi Amar,

Are your cells competent ?

Usually I produce competent cells by culture under microaerobic conditions.

I don't remember the paper from which it was elaborated but I give it to you.

In a 50 mL Falcon tube pour 20 LB with antibios.

Pick 1 colony and put in the tube.

Close the cap tightly and let grow for 12-20h. After this time open the cap in a sterile environment and let grow for 4h.

Centrifuge 3500rcf, 10 min, 4°C.

Discard the supernatant and resuspend in 600µL, 10% glycerol, 4°C.

Aliquot 40µL and freeze in liqid nitrogen or -80°C.

Competent agros prepared this manner are easily transformable with both methods, but I have better results with freeze/thaw method.

Mine is :

Let the agro tube thaw for 30 min on ice with 1µg plasmid. After this time freeze in liquid nitrogen, then put for 1 min at 37°C, and 30 min room temperature. You can do it twice or thrice. I had succes with every agro strains I tested except AGL1 and GV3350. My favorites are LBA4404 and EHA105.

Hope this helps,

Cheers

Oliv

Hi olivier, I did use them by directly taking them from overnight culture. They were supposed to be electro-competent as per manufacturer.

I suggest you to incubate for at least 6 hours (not 2 hours) after freeze thaw method. If you are doing the transformation in a 1.5 mL tube, then after 6 hours of incubation you can spin down the cells and discard the media, re-suspend in 200 ul media and then only you can culture it on plates. What I want to say is, you should increase the number of transformed cells before culturing on plates.