Triparental Mating

I. Pre-culture of bacteria:
- 2 days ago in triparental mating: Growing Agrobacterium tumefaciens LBA4404 into liquid LB + 100 ug / ml rifampicin in the dark at 280C with vigorous stirring.
- 1 day ago triparental mating: Growing E. coli helper plasmid PRK containning 2013 in liquid LB + 50 ug / ml Kanamycin AT-370C with stirring. E.coli 121 GDP Growth containning binary vector to insert DNA into liquid LB + 50 ug / ml Kanamycin AT-370C with stirring.

II. Cultivation of bacteria mixture of 3:
Centrifuge 1 ml of each bacterium at 12,000 rpm for 5-10 sec.
Discard the supernatant and add 1 ml LB and gently (to remove anti-biotic).
Repeat steps 2 to 2 times. Add to UL 300 pounds the last step.

In 3 bacteria are spread on a LB plate (agar 2%) without anti-biotics, 100 ul of each bacterium, while the surface of an LB plate is completely dry.
Culture for 1-2 days at 26-280C. After 2 days, bacteria, the form of grass.
III. Select the bacteria
Add 5 ml of 10 mm MgSO4 to the plate and sratch lawn, thus a solution to the suspension.
Prepare 4 tubes PE and placed in 900 UL 10 mm MgSO4 to each tube. Dilute the series (the original decision 10 / 1, 10 / 2, 10-3, 10 / 4)
Spread the bacteria diluted to 5 plates of LB medium + 100 ug / Rifapicine ml + 50 ng / ml Kanamycin. Cutura 2-3 days at-280C in the dark.
The culture of a single colony (single colony) in 5 ml LB + average 100 ug / ml of rifampicin + 50 ug / ml Kanamycin AT-280C for 1-2 days.
Confirm transformed by Agrobacterium PCR.
Add 15% glycerol and stored at-700C.