Team:Mexico-UNAM-IPN/Horizontal-Transfer/30 September 2008

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  1. Culture overnight of JM101 and JM109 strains.
  2. Dilution 1:100 in 30ml of sterile LB media. Incubate for 2.5-3hrs at 37°C.
  3. Thaw on ice for for 10 minutes. transfer to 12 vials.
  4. Centrifugate for 7min at 4°C at 3500rpm. Eliminate supernatant and decant. fill the vials with the culture and centrifugate again. Repeat ≈ 3 times.
  5. Resuspend the pellet in 1ml of sterile cold 10mM MgCl2in each vial. Add 500µl of 10mM MgCl2 to eeach vial.
  6. Centrifugate at 3500 rpm for 7min at 40°C.
  7. Eliminate the supernatant and resuspend in 100µl of 50mM CaCl2, Glycerol at 20%. Add 100µl of 50mM CaCl2, Glycerol at 20%. Incubate 1hr at 4°C.
  8. Transfer to 0.5ml eppendorf tubes and freeze.
  • JM101: In blue the corresponding vials to this Escherichia coli strain.
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