IGEM:Peking/2007/Count:Conjugation
From OpenWetWare
Conjugation Test
- choose the Donor cell and Recipient Cell which must be different Antibiotic.
*Day1:
- Get the plates from -4 fridge:C600-R751_psc101(Tc+), Dh5α_psb1A2(Amp+), C600_R751, Dh5α_psc101
- Amplification Culture in liquid LB for 12 hours.
*day 2:
- put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.
Donor
- Test OD600 after 30minutes of sub-culturing, stop subculturing when OD600 reached 0.45~0.6(log phase).
- After sub-culturing decant the 500uL culture into a 1.5mL tube.
- spin down (top speed for 1 min.), discard fluid.
- resuspend in 500mL LB(NO Antibiotic!), vortex.
- Repeat steps 3-4-3 in this order then progress to step 6.
- Re-suspend with 500mL LB.
- Place the cell suspension on ice.
Recipient
- decant the recipient cells(500uL culture into a 1.5mL tube) when reached the stationary phase.
- spin down (top speed for 1 min.), discard fluid.
- resuspend in 500mL LB(NO Antibiotic!), vortex.
- Repeat steps 3-4-3 in this order then progress to step 6.
- Re-suspend with 500mL LB.
- Place the cell suspension on ice.