Week 6 (07.23-07.29): Re-sequencing and re-test our original plasmids

July 23

1. Amplify bacteria of FA-7-22 for glycerol stock.

2. Miniprep plasmid of FA-7-22-2-2 and FA-7-22-2-3.

Name: FA-7-22-2-2-1 Concentration: 20.1 ng/ul A260/280: 2.19

Name: FA-7-22-2-2-2 Concentration: 15.3 ng/ul A260/280: 2.05

Name: FA-7-22-2-3-1 Concentration: 24.2 ng/ul A260/280: 1.91

Name: FA-7-22-2-3-2 Concentration: 22.3 ng/ul A260/280: 1.69

3. Amplify glycerol stock bacteria FB on plates, named FB-7-23.

4. Amplify DH10β on plate.

5. Amplify D0-7-23 plate in LB liquid medium.

6. Colony PCR of FA, FB and Co3.

7. Miniprep plasmid of Co3-7-22-1 and Co3-7-22-2 for PCR procedure.

Name: Co3-7-23-1 Concentration: 29.9 ng/ul A260/280: 1.91

Name: Co3-7-23-2 Concentration: 18.9 ng/ul A260/280: 2.00

8. Amplify Co3-7-22-1, Co3-7-22-2, Co2-7-17-1, Co2-7-17-2 and Co2-7-17-3, named Co3-7-23-1, Co3-7-23-2, Co2-7-23-1, Co2-7-23-2 and Co2-7-23-3.

July 24

1. Miniprep plasmid of D0-7-23 and Co3-7-22-2 for PCR procedure.

Name: D0-7-24-2-1 Concentration: 40.6 ng/ul A260/280: 2.00

Name: D0-7-24-2-2 Concentration: 43.7 ng/ul A260/280: 1.98

Name: D0-7-24-2-3 Concentration: 42.3 ng/ul A260/280: 2.03

Name: D0-7-24-2-4 Concentration: 51.7 ng/ul A260/280: 1.87

Name: D0-7-24-2-5 Concentration: 40.7 ng/ul A260/280: 1.87

Name: D0-7-24-2-6 Concentration: 28.9 ng/ul A260/280: 1.83

Name: FB-7-24-2-1 Concentration: 18.2 ng/ul A260/280: 2.32

Name: FB-7-24-2-4 Concentration: 16.6 ng/ul A260/280: 1.95

Name: Co-7-24-3 Concentration: 27.1 ng/ul A260/280: 1.84

Name: Co-7-24-9 Concentration: 15.3 ng/ul A260/280: 1.81

Name: Co-7-24-10 Concentration: 27.0 ng/ul A260/280: 1.78

Name: Co-7-24-12 Concentration: 15.5 ng/ul A260/280: 1.77

2. Amplify FB-7-22-2 and FB-7-23 on other plates, named FB-7-24-2 and FB-7-24.

3. Transform FA-7-23-2-3-1 and FB-7-24-2-4 into BL21*(DE3), named Co2-7-24.

4. PCR to test (1) if pCJDD0 reacts with primer FAF and FAR; (2) further confirm the success of co-transformation.

Unfortunately, we found we can achieve the same result of PCR whatever the template is if we use the same pare of primers. In contrast, we can't find there is any similarity between template and primers. We are really confused about that. It means that we can't test the success of co-transformation by PCR.

5. Amplify FB-7-23 bacteria in LB liquid medium.

July 25

Amplify Co-7-24-1-1, Co-7-24-1-2, Co-7-24-2-1 and Co-7-24-2-2, named Co-7-25-1-1, Co-7-25-1-2, Co-7-25-2-1 and Co-7-25-2-2. We prepared to induce them with IPTG later.

July 26

Transform original plasmids pCJDFA, pCJDFB and pCJDD0 into DH5α competent cells.

July 27

Peking iGEM_12 team visited us, we had a dinner together.

July 28

1. Peking and we ZJU introduced our projects this year briefly.

2. Amplify BL21(DE3)plyss strain on LB plates.

3. Transform FA-7-27-1, FA-7-27-2, FB-7-27-1 and FB-7-27-2 into DH5α competent cells respectively, named FA-7-28-1, FA-7-28-2, FB-7-28-1 and FB-7-28-2.

4. Ligation: D0 and pSB1C3, purified at July 08, named the product as K738000. Incubate at 22Degrees Celsius for 1h to increase the efficiency of transformation later.

5. Transform K738000 into DH5α competent cells, named K738000-7-28.

July 29

1. Amplify bacteria BL21(DE3)plyss in LB liquid medium for glycerol stock.

2. Amplify K738000-7-28 in LB liquid medium, named K738000-7-29.