Team:YAU-China/Notebook
1. Shake bacteria overnight
2. Transfer appropriate amount of bacteria into 40ml fresh medium .When OD600 to 0.35-0.4 are cultured by shock, the bacterial liquid is centrifuged at 4100 rpm for 5 min at 4 ℃
3. Remove supernatant, add 4 ml pre-cooled 0.1M MgCl2 suspension, ice bath for 10 min, and centrifuge at 4100 rpm for 10 min at 4℃
4. Remove supernatant, add 1.6 ml pre-cooled 0.1M CaCl2 suspension, ice bath for 30 min, and centrifuge at 4100 rpm for 10 min at 4 ℃
5. Remove supernatant, add 1.6 ml pre-cooled 0.1M glycerin CaCl2, and suspended the precipitation
6. Pack each tube into a centrifugal tube at 100 μl and store at -70 ℃ for later use
Heat shock
1. Add the ligation system or plasmid to 100 μl of competent cells and mix gently
2. Ice bath for 30 min, heat at 42 °C for 90 s immediately, and then ice bath 5 min
3. Add 1000 μl of LB medium and incubate gently at 37 °C for 1 h. Centrifuge at 5000 rpm for 5 min, remove supernatant and add 100 μl fresh LB medium, resuspend the cells, and plate
4. Place in a 37 °C incubator
Electric shock
1. Mix 10μl plasmid with 50 ml electrically-competent cells and immediately transferred to a pre-cooled 1 mm electric shock cup; electric shock cup vertical in the table tap two times, to add electric shock cup liquid will not have the existence of bubbles, with paper towel electric shock cup surface water wipe dry
2. Put the electric cup back into the ice box and take an ice bath for 5 min
3. 25 F, 1.8 kV, 200 Ω，electric shock
4. Add 1000μl liquid LB medium to electric shock cup and mix gently
5. Transfer the bacterial liquid to a 1.5 ml centrifuge tube and shake it for 1.5 h
6. Centrifuge at 4000 rpm for 5 min, discard the supernatant, add 100μl LB medium to resuspend the cells and Spread Plate
7. Place in a 37 °C incubator.
1. Culture 5 ml donor bacteria and 5 ml recipient bacteria overnight
2. Transfer 50μl in 5 ml LB medium, shake bacteria at 37 ℃ to OD600 to 0.3-0.4
3. As described above, 200 μl of recipient bacteria, 1000μl donor bacteria is taken at 1.5 ml centrifuge tube, centrifugation at 4000 rpm for 5min
4. Remove supernatant,the two precipitates were mixed and suspended with 500μl liquid LB, centrifugation at 4000 rpm for 5 min
5. Remove supernatant, the precipitate is suspended in 500 μl liquid LB medium, 4000 rpm centrifugal 5 min, abandon supernatant(repeat 2 times)
6. Suspend cells with 100 μl liquid LB, plate to the middle of the non-resistant LB plate, culture at 37 ℃ for 2 days
7. Bacteria moss is scraped into 1000 μl LB liquid medium and cells are fully suspended
8. Apply the 100μl bacteria solution on the double-resistant LB solid medium, space overnight at 37℃
9. When the conjugation is successful, the bacterial colonies will grow larger rather than smaller
1. Select single colony to shake bacteria and culture overnight
2. Transfer the seed liquid to 5 ml LB liquid and place it in a shaking table at 37 ℃, after turbidity, IPTG inducer is added into each tube and cultured in 22 ℃ shaking table for 24 h
3. Centrifuge the above liquid at a rotating speed of 8000 rmp for 3 min, and enrich the bacteria in a 2 ml centrifuge tube,200 μl PBS suspended the bacteria
4. Add steel ball to centrifugal tube, freeze and break, repeat six times
5. Centrifuge at 8000 rmp for 3 min, drain the supernatant into a new centrifuge tube, and store it at 4 ℃
1. Static culture
Take the overnight culture of PAO1 liquid, dilute with liquid LB to OD600 = 0.95, dilute liquid LB at 1:100, the diluted bacteria solution is put on a PVC plate, each process has 8 repetitions, each column is added with the corresponding supernatantand and place at 37 ℃ for 24 h
2. Sheared culture
Bacteria solution of PVC plate is sucked out, wash it with PBS twice and dry, the biofilm is stained with 0.1% crystal violet for 10 min, wash with PBS several times.Crystal violet on each biofilm of PVC plate was washed with 95% ethanol, and to determine the eluent OD570 per well
Sheared stateSingle bacterial colony of GFP-transformed PAO1 strain was selected and inoculated into LB culture solution at 37℃ and 180r/min for overnight oscillation culture. Adjust the value of 0D600 to 0.5; inoculated PAO1 solution into 24-well cell culture plates with pre-placed sterilized cover slides (8mm x 8mm as adhesive carrier), 1mL per well. 37℃for 24 h,the coverslips were gently rinsed with physiological saline to remove non-adherent cells, mounted, and the formed biofilm was observed under a fluorescence microscope
1. On the sample
The treated sample is added to the loading hole with a pipette, and a low molecular weight protein standard is added to the other loading hole for control if necessary
2. Electrophoresis
Add SDS buffer (pH8.3) to the electrophoresis tank and connect to the power supply. During the electrophoresis, concentrate the gel voltage 80 V, separate the gel voltage 120 V, electrophoresis to sds-page Loading Buffer row to the bottom of the electrophoresis tank to stop
3. Dyeing
Remove the gel from the glass plate, put it into a large petri dish, add coomassie dyeing solution, and shake the table for dyeing for 1 h at room temperature
4. Decolorizing
After dyeing, pour out coomassie dye and recycle it. Rinse the gel with clean water, then add the decolorization solution and decolorize the gel several times until the protein band is clearly visibledecolorizing
1. Install the gel tank and insert the appropriate comb
2. Take appropriate amount of agarose in the triangle bottle, add appropriate amount of TAE buffer, and heat in the microwave until completely dissolved
3. When it is not very hot, add appropriate amount of nucleic acid dye, pour the solution into the gel tank, stand for 30 min at room temperature, then cool and solidify
4. Pull out the comb vertically after solidification. Remove the gel together with the gel sheet. Clean the gel fragments under the gel sheet and in the gel tank. Put the gel into the electrophoresis tank
5. Add the sample to the orifice with a pipette
6. Adjust the voltage to 120 V, and power off after about 30 min
7. Put the gel on the ultraviolet transmission detector and observe through the observation hole
8. Put the observed gel back into the corresponding recycling box
In the course of the experiment, we were divided into three groups, which were closely linked and the experiments were closely related. We ensure the test record of each group presented as complete as possible. We are willing to accept good suggestions to improve and optimize our experiments.