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

=Lock & Key By Yu Tao=

Acknowledgement and Farewell
=Tandem OriT by Mingzhi Qu & Ze Ren=
 * 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.

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:
 * 1) R751+psc101   0.510
 * 2) C600_R751     0.470
 * 3) C600_psc101   0.480

Recipient

 * final OD600:
 * 1) 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               /          /