IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-8
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OriT by Qu Mingzhi
Conjugation Test:R751_pSC101 X Dh5α_psb1A2
- Donor: C600-R751_psc101 (Tc+)
- Recipient: Dh5α+ psb1A2 (Amp+)
- Control:
- donnor:C600_R751
- donnor:Dh5α_psc101
- mixing condition: 220rpm vs. 60rpm; 60min vs. 120min
*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.