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.
 * Notes: Some controls may growth on double antibiotic plate when these cultures are not being diluted.

result

 * The frequency of transfer was expressed as the ratio (percentage) of the number of transconjugants to that of the donors.