I want to insert my 14kb recombinant plasmid into A.Rhizogenous, what should I do during my transformation. I transformed my plasmid with freeze/thaw transformation method, but it didn't work. I'm looking for freeze/thaw transformation method or any other one which anyone used successfully. Thank you for the answer.
Thank you for your answer. I would like to know the heat-shock method, step by step if it's possible.
Yeah, you are right. But could you please send me step-by-step of this transformation method.
In our hands, electroporation is the most effective transformation for any agrobacterium. You will need to wash extensively your cells (in 10% cold glycerol), to avoid the presence of any salts before electroporation. Then, electroporation itself depends on the apparatus and cubette you used.
Josefa
Just as Josefa said, clean the bugs with sterile 10% (w/v) glycerol (cold) 1 or 2 times, then freeze in 10% w/v glycerol (I generally get 40 aliquots of 100 uL out of 20 mL culture, and these are very heavy aliquots, I should probably split these down by more than 1/2, but whatever). Thaw on ice, add plasmid (I generally use between 20 to 100 ng, which is probably way more than is necessary, but again, we are cooking here, not baking. Zap those suckers in an ice-chilled 1 mm gap electroporation cuvette, 1.8 kV, 400 Ω. Hopefully you don't have arcing (which, if you cleaned the cells well enough, you won't), then slowly deliver 1 mL of LB (or whatever liquid medium your bug prefers), gently mix, then withdraw the mixture from the cuvette to a sterile tube. Recover with gentle shaking at 28 degrees C for 1 hour, then plate (I usually do 2 plates, 1 with 50 uL, the other with the remainder (which I pellet by centrifugation for 1 minute, then resuspend in whatever residual liquid medium remains on the tube walls after gentle decanting ~100 to 200 uL) on the solid medium of choice supplemented with the appropriate antibiotics to select for the binary vector. Probably 2 to 3 days later you will have what you need.
Dear Josefa M. Alamillo and Ray Collier, I really appreciate your complete answers and Thank you.
Try this protocol
Storage of competent cells for Agrobacterium transformation by Rainer Hofgen and Lothar Willmitzer. I have attached the paper below. Hope you will find it helpful.
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