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

 * According to the conjugation protocol
 * final OD600:
 * 1) C600_R751+psc101   1.750
 * 2) C600_R751          2.274
 * 3) Dh5α+psc101   1.883

Recipient

 * 1) According to the conjugation protocol
 * final OD:
 * 1) Dh5α+pSB1A2   2.066

mix condition
No  Donor                          rpm        time(min) 1   R751+psc101_stationary         220        120 2   R751+psc101_stationary         220        60 3   R751+psc101_stationary         60         120 4   R751+psc101_log                220        120 5   R751                           220        120 6   Dh5α+psc101                    220        120

Plating the conjugant mix

 * All mix were set up serial dilutions:original, 10-1, 10-2.
 * culture on Tc+/Amp+ plate.

result
No Donor                  X   recipient  time rpm  clones(original)  clones(e-1)   clones(e-2) 1 R751+psc101_stationary  X  Dh5α_pSB1A2  120  220     2000+              300+        <5 2 R751+psc101_stationary  X  Dh5α_pSB1A2   60  220     2000+              200+        <5 3 R751+psc101_stationary  X  Dh5α_pSB1A2  120  100     2000+              500+        <20 4 R751+psc101_log         X  Dh5α_pSB1A2               2000+              <100        <10 5 R751                    X  Dh5α_pSB1A2  120  220     50+                 <10        <3 6 Dh5α+psc101             X  Dh5α_pSB1A2  120  220    2000+               200+        0
 * NEXT day:
 * test failed because control received clones....they thould not grown on Tc+/Amp+ plate!!

Amplification Culture of OriT_J23066_OriT_pSB1A2(normal T4 ligase)

 * select Positive OriT_J23066_OriT_pSB1A2 Colonies from Plate, Culture in liquid LB,waiting for mini-prep.

=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:
 * 1) marker
 * 1) 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.