IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-12

=Tandem OriT by Mingzhi Qu, Ze Ren=

Conjugation Test:R751_pSC101 X Dh5α_psb1A2(II)

 * Donor:    C600-R751_psc101 (Tc+)
 * Recipient: Dh5α+ psb1A2 (Amp+)
 * Control:
 * 1) donnor：C600_R751
 * 2) donnor：C600_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   0.505
 * 2) C600_R751     0.508
 * 3) C600_psc101   0.570

Recipient

 * final OD600:
 * 1) Dh5α+pSB1A2   1.571

Plating the conjugant mix

 * All mix 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 C600_R751_psc101_log    X  Dh5α_pSB1A2  90  220     1000+               100+       <10 2 C600_R751               X  Dh5α_pSB1A2  90  220       0                  0          0 3 C600_psc101             X  Dh5α_pSB1A2  90  220      100                 20         0

=oriT Knock Out by Liu Ting=

PCR system(Every 200 ul is distributed to 10 tubes, that is, 20ul/PCR tube)
pKO3-R(100uM) 2  ul R1S(20uM)      10 ul dNTP(2.5uM)    4  ul 10X Taq Buffer 20 ul Taq            1  ul ddH2O          163ul (template)
 * PCR system for DH5a-oriT-deleted R751

Total         200ul F1S(20uM)     10 ul F2A(20uM)      10 ul dNTP(2.5uM)    4  ul 10X Taq Buffer 20 ul Taq            1  ul ddH2O          155ul (template)
 * PCR system for DH5a-oriT-deleted F

Total         200ul S1S(100uM)    2  ul S2A(100uM)     2  ul dNTP(2.5uM)    4  ul 10X Taq Buffer 20 ul Taq            1  ul ddH2O          171ul (template)
 * PCR system for DH5a-pUC18 with oriT-deleted pSC101 fragment

Total         200ul

PCR condition
for all the above three: 94℃ 10min; (94℃ 45s,51℃ 1min,72℃ 1min30s)/cycle X 30 cycles; 72℃ 10min; 16℃ 10min; end.

electrophorsis result
none...

=Lock & Key By Yu Tao=

Transformation Result: R0040<-J01010 and R0010<-J01008

 * There are colonies both in the experimental plate and the negative control plate.
 * For R0040<-J01010, more clonies grow in the expeimental plate, but the opposite result for R0010<-J01008.
 * Number of colonies: around 100 or 200.
 * Select probable positive colonies from the experimental plate, culture them in liquid LB overnight for mini-prep.

Preparation of Competent Cells

 * Because the newly produced competent cells are of low efficiency, I decide to make some competent cells once more.
 * Transform 1uL B0015 competent cells for efficiency test.