IGEM:Peking/2007/Count:Conjugation

Conjugation Test

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

Day1:

1. Get the plates from -4 fridge：Donor, Recipient, Control.
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 30 minutes 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.

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, 10-3, 10-4, 10-5
• 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.

see also

IGEM:UC_Berkeley/2006/Conjugation