Week 8 (08.06-08.12):

August 06

1. Amplify Venus-8-2 in LB liquid medium. Add 0mM, 0.5mM, 1mM, 1.5mM theophylline accordingly.

2. PCR of part K738001:

3. PCR of original pCJDFA and pCJDFB.

4. Amplify FAM-8-5 and FBP-8-5 for minipreparing plasmids.

Name: FAM-8-6-1 Concentration: 56.7 ng/ul A260/280: 1.95

Name: FAM-8-6-2 Concentration: 57.3 ng/ul A260/280: 1.95

Name: FAM-8-6-3 Concentration: 86.8 ng/ul A260/280: 2.06

Name: FBP-8-6-1 Concentration: 21.0 ng/ul A260/280: 2.14

Name: FBP-8-6-2 Concentration: 15.5 ng/ul A260/280: 2.14

Name: FBP-8-6-3 Concentration: 15.6 ng/ul A260/280: 2.07

5. Add theophylline 0.08g/10mL into Venus-8-6, observe through fluorescence microscope.

6. Purify PCR product of K738001, named K738001-8-6.

7. Co-transform FA-8-5-3, FB-8-5-2 and D0-4 into BL21*(DE3), named Co3-8-6. Co-transform FA-8-5-3 and FB-8-5-2 into BL21*(DE3), named Co2-8-6.

August 07

1. Double digest K738001-8-6 with Xba1 and Spe1. Purify the product before ligation step.

2. Ligation: K738001-8-6 and pSB1C3.

3. Transform the product of ligation into DH5α competent cells.

August 08

Pick up single colonies from plate of K738001-8-7 and amplify it in LB liquid medium and LB plate.

August 09

1. Amplify Co2-8-7 and Co3-8-7 in LB liquid medium, induce with IPTG when OD600 is about 0.6 (mid log stage).

2. Miniprep plasmids of K738001-8-8:

Name: K738001-8-9-1 Concentration: 174.2 ng/ul A260/280: 1.91

Name: K738001-8-9-2 Concentration: 298.5 ng/ul A260/280: 1.87

3. Amplify FBP overnight for minipreparing plasmids later.

4. Mutation of D0:

PCR1: delete 3bp after MS2 in D0.

PCR2: delete 3bp before MS2 in D0.

August 10

1. Amplify Co2-8-7 and Co3-8-7 in LB liquid medium. Induce with IPTG when OD600 is about 0.6 (mid log stage). Take photos with fluorescence microscope.

2. Miniprep plasmids of FB-8-9.

Name: FB-8-10-1 Concentration: 14.6 ng/ul A260/280: 1.97

Name: FB-8-10-2 Concentration: 24.4 ng/ul A260/280: 1.93

3. Mutation of D0:

PCR1: PCR with primer P1 and P2. Set a temperature gradient from 44Degrees Celsius to 54Degrees Celsius.

PCR2: product of 47.5Degrees Celsius from PCR1 20 ul + Taq Mix 37.5 ul + D0RLX 1.5 ul + ddH2O 36 ul.

PCR3: 49.6Degrees Celsius product 20 ul + 10 ul Taq Mix +0.5 ul D0RLX + 9.5 ul ddH2O.

4. Amplify FB-8-7 in LB liquid medium.

August 11

1. Miniprep plasmids of FB-8-11.

Name: FB-8-11-1 Concentration: 33.9 ng/ul A260/280: 2.06

Name: FB-8-11-2 Concentration: 50.2 ng/ul A260/280: 2.04

2. Amplify Co2-8-7 and Co3-8-7 in LB liquid medium. Induce with IPTG when OD600 is about 0.6 (mid log stage). Test fluorescence strength with hybrid synergy reader. Exciting light 535nm, absorbing light 480nm.

August 12