Transformation of clones in Agrobacterium tumefaciens by electroporation
(The performed protocol using earlier described electro-competent cells of Agrobacterium tumefaciens)
Only 2 ml of each clone was used for electro-transformation by electro cell manipulator 600 (BTX San Diego, California). For electroporation the following protocol was used.
1. Electroporation cuvettes 1 mm gap were placed on ice. Viols of frozen electro-competent cells of Agrobacterium were allowed to thaw on ice.
2. 1 mg DNA of the recombinant plasmid was mixed with 50 ml of electro-competent cells in the electroporation cuvettes on ice.
3. The condition for electroporation were set as recommended by the manufacturer:
Choose mode T 2.5 KV
Set resistance R R5 (129ohm)
Set charging voltage 1.44 KV
4. The electro-competent cells containing the DNA mixture were transferred to electroporation cuvette.
5. Pulse was given and 1 ml of liquid LB medium was added immediately, mixed gently and transferred to a 1.5 ml eppendorf tube and incubated at 28°C for 1 hour with vigorous shaking.
6. 200 μl and 400 μl of transformed culture were spread on petri plates containing solid LB medium supplemented with 50 µg of rifampicin and 50 µg of kanamycin/ml, so that only transformed cells should multiply.
7. When the liquid was absorbed completely the plates were sealed with sealing film and kept at 28 0C for 2-3 days.
8. At the end of incubation colonies were picked with sterile toothpicks and cultured in 5ml liquid LB medium in 50 ml tube containing 50 µg of rifampicin and 50 µg of kanamycin /ml.
9. Culture tubes were kept at 28 0C on shaker in Agrobacterium growth room for 48 hours with vigorous shaking.
Transformants were confirmed through PCR.
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