IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-27

=Tandem OriT by Mingzhi Qu, Qingsi Zhen=

Conjugation Test:F+_F-OriT-pSB1A2 X Dh5α_psb3K3(II)

 * Donor:    F+_F-OriT-pSB1A2 (Amp+)
 * Recipient: Dh5α_psb3K3(Kan+)
 * Control:
 * 1) donnor：F+
 * 2) donnor：Dh5α_OriT-pSB1A2 (Amp+)
 * mixing condition: 220rpm, 90min.


 * Day1:
 * 1) Get the plates from -4 fridge：F+_F-OriT-pSB1A2 (Amp+), F+, Dh5α_OriT-pSB1A2, Dh5α_psb3K3(Kan+)#Amplification Culture in liquid LB for 12 hours.
 * Day2:
 * put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.

Donor
F+_F-OriT-pSB1A2 0.588 F+               0.522 Dh5α_OriT-pSB1A2 0.477
 * final OD600:

Recipient

 * final OD600:
 * 1) Dh5α_psb3K3      1.760

Mixing

 * Wash 2 times.
 * mix with Lb- as control.

Plating the conjugant mix

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

result(Next day)
No Donor                  X   recipient  time rpm  clones(original)  clones(e-1)   clones(e-2) 1 F+_F-OriT-pSB1A2        X  Dh5α_psb3K3  90  220      full               2000+       300+ 2 F+_F+_F-OriT-pSB1A2     X  Lb-          90  220        /                 /           / 3 F+                      X  Dh5α_psb3K3  90  220        /                 /           / 4 F+                      X  Lb-          90  220        /                 /           / 5 Dh5α_OriT-pSB1A2        X  Dh5α_psb3K3  90  220      full              1000+        <100 6 Dh5α_OriT-pSB1A2        X  Lb-          90  220        /                 /           /