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

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               /          /