IGEM:Peking/2007/Count:Conjugation
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Conjugation Test
- choose the Donor cell and Recipient Cell which must be different Antibiotic.
Day1:
- Get the plates from -4 fridge:Donor, Recipient, Control.
- 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 30 minutes 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.
mixing
- Donor cells(log phase, include control) mixed with recipient cells(stationary phase).
- Mix by vortexing.
- culture in 37℃ for 90 minutes, 220rpm.
Plating
- Setting up serial dilutions:original, 10-1, 10-2.
- Pipette 200µL of each serial dilution onto an Double antibiotic plate.
result
- The frequency of transfer was expressed as the ratio (percentage) of the number of transconjugants to that of the donors.