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:

#R751+psc101    0.505
#C600_R751      0.508
#C600_psc101    0.570

Recipient

• final OD600:

#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

colony PCR test for DH5a-oriT-deleted R751 , DH5a-oriT-deleted F & DH5a-pUC18 with oriT-deleted pSC101 fragment

PCR system(Every 200 ul is distributed to 10 tubes, that is, 20ul/PCR tube)

• PCR system for DH5a-oriT-deleted R751

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)
--------------------
Total          200ul

• PCR system for DH5a-oriT-deleted F

F1S(20uM)      10 ul
F2A(20uM)      10 ul
dNTP(2.5uM)    4  ul
10X Taq Buffer 20 ul
Taq            1  ul
ddH2O          155ul
(template)
--------------------
Total          200ul

• PCR system for DH5a-pUC18 with oriT-deleted pSC101 fragment

S1S(100uM)     2  ul
S2A(100uM)     2  ul
dNTP(2.5uM)    4  ul
10X Taq Buffer 20 ul
Taq            1  ul
ddH2O          171ul
(template)
--------------------
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: R0040<-J01010 and R0010<-J01008

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.