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

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Contents

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

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

Recipient

  • final OD600:
#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        /                 /           /
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