Jonesma Lab:Protocols:Agrotransform

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Transforming Agrobacteria with cold-shock transformation)

  1. Thaw competent Agrobacterium on ice (use 250 ul per transformation reaction) and add DNA (1-5 ul of CsCl-purified DNA or 10 ul of standard E. coli miniprep DNA).
  2. Incubate the mixture on ice for 5 minutes
  3. Transfer mixture to liquid nitrogen and incubate for 5 minutes.
  4. Incubate the mixture for a further 5 minutes in a 37 degree C water bath.
  5. Add 1 ml of LB to each tube, seal well, and place the tubes on a rocking table for 2-4 hours at room temperature.
  6. Collect the cells by spinning briefly in a microcentrifuge and spread them on two LB agar plates containing the appropriate antibiotic. Include the antibiotic for the T-DNA vector.
  7. Incubate cells for 2 days at 28 degrees C.