IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-8

OriT by Qu Mingzhi

Conjugation Test:R751_pSC101 X Dh5α_psb1A2

• Donor: C600-R751_psc101 (Tc+)
• Recipient: Dh5α+ psb1A2 (Amp+)
• Control:

1. donnor：C600_R751
2. donnor：Dh5α_psc101

• mixing condition: 220rpm vs. 60rpm; 60min vs. 120min

*Day1:

1. Get the plates from -4 fridge：C600-R751_psc101(Tc+), Dh5α_psb1A2(Amp+), C600_R751, Dh5α_psc101
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 30minutes 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.

Lock & Key by Yu Tao

PCR R0010<-J23066

• vr and vf2 primer (standard sequencing primer): stored as 100uM.
• PCR system contains:

0.5 µL    Template
1 µL      Primer pf1
1 µL      Primer pr1
4 µL      dNTP
0.5 µL    Taq
5µL       10 X buffer
38µL      ddH20
--------------------------
50 µl      Total

• Primer final concentration 1uM.
• PCR program setting:

Step1     94℃ 5min
Step2     94℃ 30s 
Step3     60℃/62℃ 30s
Step4     72℃ 90s 
Step5     Go to step 2 for 29 times 
Step6     72℃ 10min
End

Electrophorsis Result

• from left to right:

2. marker

Key1 & Lock1 Efficiency Test

• Preculture the following 4 groups of DH5a overnight.

1. I7100-DH5a
2. R0010.J23066(Key)-DH5a
3. R0010.J23066(Key)+R0040.J23078.E0040.B0015(Lock) -DH5a
4. DH5a negative control.