Team:Xiamen City/Notebook
6.19
1. understand how HPLC and PCR works2. learn to use pipettes and get familiar with the lab
3. cultivation of E.coli Get three Erlenmeyer flasks each with 200μl E.coli and 300 ml LB culture medium and put three of them into on the shaking table for 1 day at 37℃
6.20
1. measure the OD(optical density) of the bacteria2. Construct of the System of Adaptive Evolution of E.coli
Put five kinds of solutions on the shaking table at 37℃ to provide a normal surviving environment for E.coli
6.21
1. measure the OD of E.coli
2.continue cultivation of the system of adaptive evolution of E.coli
6.22
1. measure the OD of E.coli
2.continue cultivation of the system of adaptive evolution of E.coli
3. amplify segments of DNA via PCR (polymerase chain reaction)
3.1 solutions
3.2 mode
The first PCR failed to provide the specimen because the fragment length of DNA did not reach the expectation.
6.23
1. measure the OD of E.coli
2. proceed PCR again
3.1 solutions
3.2 mode
The EP tube exploded in the thermal cycler and the solutions was contaminated.
8.11
Plasmid Extraction
1.Take 1-4mL bacterial liquid cultured in LB medium,
2.Centrifuge of the solution for 1 min at 12000*g, which separates the clumped cellular debris from the DNA.
3.Discard the supernatant
4.Add 250 μL S1 Buffer to suspend the bacterial precipitation (the suspension should be uniform and no small bacteria should be left).
55.Add 250 μL S2 Buffer, gently and fully flip the solution up and down 4-6 times until the bacteria are fully broken down and a transparent solution is formed.
6.Add 250 μL S3 Buffer, gently and fully flip the solution up and down 4-6 times.
7.Centrifuge of the solution for 1 min at 12000*g.
(Purification of plasmid DNA by centrifugation)
8.Transfer the supernatant left after centrifugation to the preparation tube.
9.Centrifuge the solution for 1 min at 12000 * g, then discard the filtrate.
10.Put the preparation tube back into the centrifuge tube, add 500 μL W1 Buffer, centrifuge the solution for 1 min at 12000 * g, and then discard the filtrate.
11.Put the preparation tube back into the centrifuge tube, add 700 μL W2 Buffer, centrifuge the solution for 1 min at 12000 * g, and then discard the filtrate.
12.Put the preparation tube back into the 2mL centrifuge tube, add 60 μL Eluent or deionized water to the center of the tube membrane, put the solution at room temperature for 1 min, and centrifuge it at 12000 * g for 1 min.
Transfer
1.Take several centrifuge tubes and add to 2mL KAN medium respectively.
2.Take the cultured psB1K3-RBS-A-GFP, psB1K3-RBS-B-GFP, psB1K3-RBS-D-GFP, psB1K3-GFP, psB1K3-Facas12a, psB1K3-LacZ, psB1K3-J23100-B0034-ter, use the needle tip to take a small amount, and put it onto the shaking table.
8.13
Transfer
1.Take several test tubes and add to 2mL KAN medium respectively.
2.Take the cultured psB1K3-RBS-A-GFP, psB1K3-RBS-B-GFP, psB1K3-RBS-D-GFP, psB1K3-GFP, psB1K3-Facas12a, psB1K3-LacZ, psB1K3-J23100-B0034-ter, use the needle tip to take a small amount, and put it onto the shaking table.
PCR
1.Put chemicals below to the centrifuge tube.
2.Adjust the reaction procedure, centrifuge the mixture, and immediately place it on the PCR machine for amplification. The reaction process is predenatured at 93 ℃ for 3 min, and then enter the cycle amplification stage: 93℃ 40s → 58℃ 30s → 72℃ 60s, the cycle repeats it for 30-35 times, and finally keep at 72 ℃ for 7 min.
Protein Electrophoresis
1.Add 24 μL psB1K3-LacZ, psB1K3-GFP, psB1K3-RBS-A-GFP, psB1K3-RBS-B-GFP, psB1K3-RBS-D-GFP, MG1655 bpul, DH10B bpul, BL21KDE33 to 1.5mL tube respectively, and then add 6 μL loading.
2.Add 500 μL deionized water and centrifuge the tubes respectively.
3.Add another 500 μL deionized water, blow and suspend the solution.
4.Put the tubes 99℃ water for 15 minutes.
5.After heating, centrifuge the tubes for 5min.
6.Take and install the gel, add the electrophoresis buffer to the sample hole, and check if there is any leakage. Add marker, to the first and last sample holes, add psB1K3-LacZ,psB1K3-GFP and psB1K3-RBS-A-GFP, psB1K3-RBS-B-GFP, psB1K3-RBS-D-GFP, MG1655 bpul, DH10B bpul, BL21KDE33 to the other sample holes, respectively.
7.Connect the electrophoresis device to the power supply. Connect the positive electrode to the tank and the negative electrode to the slot for electrophoresis. The voltage is adjusted to 160V.
8.Turn off the power supply and disconnect the electrode until the bromophenol blue reaches the bottom of the release adhesive. Remove the glass sheet from the electrophoresis device and then remove the gel.
9.Soak the gel in Coomassie brilliant blue dye and dye it in a horizontal shaking bed for 15 min.
10.Observe the protein bands
Tests of bpul
the solution of m-phenylenediamine is added to the bacteria (MG1655, BL21, and DH10B expression strain). Place the solutions onto the shaking table.The dimmer color of each tube of solution indicates that the degradation was successful.