Team:QHFZ-China/Experiments
Experiments
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Though the COVID-19 affect our experimental process, we still feel fortunate to have the change doing our experiment instead of just gathering online. In this page, we'll show the protocol of the experiments we do this year and also the process on how we do the lyophilization.
Molecular Cloning
【1】PCR
Primers were designed by snapgene software and synthesized at GENEWIZ Company. PCR was performed by following the manuals of PrimeSTAR Max Premix (2X) (Taraka, R045) or 2 X EasyTaq PCR SuperMix (+ dye) (Transgene, M256). The former was used for construction and the latter was used for identification.
【2】Gel extraction
DNA fragment were separated by agarose gel electrophoresis and then recycled by TIANgeo Midi Purification Kit (TIANGEN, DP209-02) or AxyPrep DNA gel Extraction Kit (AXYGEN, AP-GX-250).
【3】Gibson
Gibson was performed by following the manual of pEASY- Basic Seamless Cloning and Assembly Kit (Transgene, CU201).
【4】Endonuclease digestion and ligation
The endonucleases and T4 ligase were bought from NEB Company and the experiments were performed by following instructions.
【5】Transformation
Transformation was performed by following the manuals of Trans5α Chemically Competent Cell (Transgene, CD201) or Trans1-T1 Phage Resistant Chemically Competent Cell (Transgene, CD501).
【6】Plasmid extraction
Bacteria were cultured in lysogeny broth (LB) at 37℃ overnight and plasmid extraction was performed by following the manuals of TIANprep Mini Plasmid Kit (TIANGEN, DP103-02) or AxyPrep Plasmid Miniprep Kit (AXYGEN, AP-MN-P-250).
【7】Sequencing
Sanger sequencing was done by GENEWIZ Company and snapgene software is used to align the sequences.
Lyophilization Process
【Day 1】Induction culture
(1) Pick clones which are in good condition and put them into 500 μL LB medium containing antibiotics. Shake them to grow at 37℃ for 5~7 hours until the bacteria solution becomes turbid.
(2) Add 2 mM iPTG into 3 mL LB medium containing antibiotics. Add 3 μL of the bacteria solution mentioned in step 1 to dilute the bacteria by the ratio of 1:1000. Shake the solution to grow the bacteria at 37℃ overnight.
【Day 2】Freeze-dried
(1) If fluorescence induced by the iPTG is detectable in the control group (GFP), continue conducting the experiment.
(2) Use spectrophotometer to measure the OD600 of the bacteria solution, OD600 = 1 equals to 109 cells. If the OD600 value is between 0.1 and 1, There is a linear relationship between OD600 and bacterial density. Calculate the volume of bacterial solution for 109 cells by using the formula V = 100 / (OD600 × Dilution ratio).
(3) Take out a measured amount of 109 cells and centrifuge it at 8000 rpm for 3 min. Then pour out the supernatant.
(4) Resuspend the bacteria in a 15 mL tube with pre-refrigerated 100 μL 3% glucose solution.
(5) Take off the cover of the tube and put the bacteria into the cold trap. Open the compressor of the lyophilization machine and freeze the shake tube for 2 h at -70℃.
(6) Put the caky bacteria solution into the drying chamber of the lyophilization machine. Open the vacuum pump to dry it in vacuum for 6h at 1 Pa vacuum degree.
(7) Turn off the vacuum pump, place it at seal box filled with silica-gel desiccant a for 2 days at room temperature.
【Day 3】Room temperature storage
【Day 4】Detect the survival rate
(1) Add 1 mL of sterile water to the tube, vortex for 15 s, placed it at room temperature for 10 min.
(2) Adjust the density of the bacteria solution by gradient dilution, then spread 100 μL of the bacteria solution on the LB plate.
(3) If the density above is not suitable, take 100μL of the solution and spread it on the LB plate after several gradient dilutions.
(4) Culture the bacteria overnight at 37℃.
【Day 5】Cell Count
(1) Take out the LB plate and take photos to record experimental results.
(2) Use the automatic cell counting function of Image J to count the colone number on the LB plate, then compare the results between each group.
Protein
SDS-PAGE
Bacteria are cultured overnight. OD600 is tested. The same nubmer
(3~5 x 10e9) bacteria of different group is collected. Then the cells are lysed by 100~200 μL 4% SDS for 5 min in room temperature, then 10 min at 95℃. Then 6x loading is added. The SDS-PAGE was carried with a 12% precast polyacrylamide gel. Coomassie bright blue stain is used to observa the bands.
Cytology experiments
【1】Cell culture
Human HeLa cells were cultured in complete DMEM (CD) medium (89% High-glucose DMEM, 10% fetal bovine serum (FBS) and 1% Penicillin / streptomycin). Generally, the medium was changed every day. Tyrisin was used for passage. Usually, 1 × 105 cells were seeded in every well of 6-well plate.
【2】Transient transfection
Plasmid was extracted by TIANprep Mini Plasmid Kit (TIANGEN, DP103-02). 1 × 105 HeLa cells were seeded in a well of 24-well plate. 24 h later, transient transfection was performed by following the manual of Lipofectamine™ 3000 Transfection Reagent (Invitrogen, L3000001). The total amount of plasmids in different wells were kept the same by adding pcDNA3.1-eGFP as the no-treatment control group and added pcDNA3.1-Dsup as the experimental group.
【3】After transfection of Dsup for 48 hours, take photos to record the cell cellular state or fluorescence intensity. At the same time, irradiate the cells with ultraviolet light for 15 s. Then placed them back to the CO2 incubator. Culture the cells for another 48 h. Then fix the cells by 4% PFA for 30 min.
【4】Observe the survival rate by using crystal violet staining methods and Image J software to calculate the intergrated density.
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