IGEM:Peking/2007/Count:Conjugation

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Contents

Conjugation Test

  • choose the Donor cell and Recipient Cell which must be different Antibiotic.

*Day1:

  1. Get the plates from -4 fridge:C600-R751_psc101(Tc+), Dh5α_psb1A2(Amp+), C600_R751, Dh5α_psc101
  2. Amplification Culture in liquid LB for 12 hours.

*day 2:

  1. put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.

Donor

  1. Test OD600 after 30minutes of sub-culturing, stop subculturing when OD600 reached 0.45~0.6(log phase).
  2. After sub-culturing decant the 500uL culture into a 1.5mL tube.
  3. spin down (top speed for 1 min.), discard fluid.
  4. resuspend in 500mL LB(NO Antibiotic!), vortex.
  5. Repeat steps 3-4-3 in this order then progress to step 6.
  6. Re-suspend with 500mL LB.
  7. Place the cell suspension on ice.

Recipient

  1. decant the recipient cells(500uL culture into a 1.5mL tube) when reached the stationary phase.
  2. spin down (top speed for 1 min.), discard fluid.
  3. resuspend in 500mL LB(NO Antibiotic!), vortex.
  4. Repeat steps 3-4-3 in this order then progress to step 6.
  5. Re-suspend with 500mL LB.
  6. Place the cell suspension on ice.

Plating the conjugant mix

result

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