IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-23

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Lock & Key By Yu Tao

Acknowledgement and Farewell

  • First, I have to say thank you to Mingzhi because he helped me with almost all the experiments these ten days so that I could spend most of my time accompanying my girlfriend. n_n Thank you Mingzhi.
  • Second, I am going home today and I am very excited. Goodbye and Work hard, my friends. I like the feeling of home, Ah Ha! :) I will be back on Sep, 1st.

Tandem OriT by Mingzhi Qu & Ze Ren

Conjugation Test:R751_pSC101 X Dh5α_psb1A2(III)

  • Donor: C600-R751_psc101 (Tc+)
  • Recipient: Dh5α+ psb1A2 (Amp+)
  • Control:
  1. donnor:C600_R751
  2. donnor:DH5α_pSC101
  • mixing condition: 220rpm, 90min.
  • Day1:
  1. Get the plates from -4 fridge:C600-R751_psc101(Tc+), C600_psb1A2(Amp+), C600_R751, Dh5α_psc101
  2. Amplification Culture in liquid LB for 12 hours.
  • Day2:
  • put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.

Donor

  • final OD600:
#R751+psc101    0.510
#C600_R751      0.470
#C600_psc101    0.480

Recipient

  • final OD600:
#Dh5α+pSB1A2    1.631

Plating the conjugant mix

  • C600_R751_psc101 group was 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  C600_R751_psc101        X  Dh5α_pSB1A2  90  220      full              1000+       <10
2  C600_R751               X  Dh5α_pSB1A2  90  220       40+                /          /
3  C600_psc101             X  Dh5α_pSB1A2  90  220       full               /          /   
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