Plasmid mini extraction
1. Heat shock transformation of Escherichia coli
2. Mini shaking of the plasmid
3. Plasmid mini extraction
Plasmid midi extraction
1. Heat shock transformation of Escherichia coli
2. Midi shaking of the plasmid
3. Plasmid midi extraction
Cell rhythm detection
Reverse transcription
1. Degenerate RNA template.
2. Configure the 1st strand cDNA synthesis reaction solution.
3. Synthesize the 1st strand cDNA according to the following conditions.
PCR
1. PCR system
2. PCR process
3. Recovery scheme of PCR reaction solution
Homologous recombination
(pEASY®-Basic Seamless Cloning and AssemblyKit)
1. Preparation of vectors and target fragments
2. Establishment of cloning reaction system
3. Transform
4. Positive clone detection
Restriction enzymes cutting analysis of positive clones
Sequencing
Enzyme cutting and connecting
1. Enzyme cutting system
2. Connecting
Transfection
(NeofectTM DNA transfection reagent)
1. Cell paving board
Cells were planted in six-well plates one day in advance with a cell density of 60% -80% for transfection.
2. The transfection process
Transfection
(Reagent/LipofectamineTM 3000)
Experiments on verification of signaling pathways through calcium flow signal changes
1. Coelenterazine (Coelenterazine-h) pretreat the cells
For cells in each dish transfected with the plasmid, follow the following steps:
2. Collect the cells
3. Configure the solution required
4. Equilibrate the cell suspension
5. Sirius Single-tube-type chemiluminescence detector test the Changes in the Ca2+ in cells.
(1) GnRH recombinant protein was diluted in 10 concentration gradients in a 1:3 gradient in serum-free medium, with the highest concentration of 10 µM (take 15 µL).
GnRH (10 mM) storage solution configuration: 1.1 mg GnRH powder + 104 mL ddH2O, divided into 15 µL per tube, preserve in -80℃.
(2) Turn on the power supply of the Sirius single-tube chemiluminescence detector and set it up:
The test intracellular Ca2+ concentration parameter was set as:
After the instrument door was closed, 90 µL of cell suspension was injected from the INJECTOR1 into the plastic tube for 1 s. After 35 s, 50 µL of 1% Triton X-100 was injected from the INJECTOR2 to the plastic tube at 0.2 s for 225 times, and the total detection time was 45 s.
(3) Set up the FB12 Sirius software.
(4) Cell activity detection:
Take the disposable plastic test tube, add 5 µL ATP (2 µm) to the bottom of the tube, and insert the test tube for testing.
(5) Determine the change of Ca2+ concentration in cells.
If ATP can stimulate the increase of Ca2+ concentration in cells, 10 µL of the highest concentration GnRH standard was added to the bottom of the plastic tube into the Sirius single tube chemiluminescence detector, and the whole detection frame was pushed into the detector to wait for the automatic detection of the instrument, and each concentration was repeated for three times. After the sample is tested, replace the test tube and continue to test the effect of the next GnRH standard concentration on the intracellular Ca2+ concentration. The centrifuge tube with cell suspension should be gently shaken to avoid cell deposition at the bottom of the tube. When replacing different cell suspensions, rinse pipe 1 twice with DI water before flushing the pipe with new cell suspension to prevent mixed effects of different types of cells.
(6) After the test, save the data and clean the two pipes according to the instrument operation guide.
Western blot
1. Prepare the cell lysate firstly (2%SDS protein lysate : Buffer/ protease inhibitor / phosphatase inhibitor = 50:1).
2. Suck off the old culture in the cell petri dish or orifice plate, add the 2%SDS cell lysate of 100ul according to the cell density and quantity. Rest on the ice for a while.After the cells fully cleaved, scratch them with the head of the gun, and finally transfer the lytic cells to the named EP tubes. And then denature the protein. According to the properties of the protein, heat the protein on a dry calorimeter at 95 ℃ for 10 minutes. After denaturation, the protein is ultrasonic on the cell ultrasonic breaker.
3. The denatured ultrasonic protein was quantified by BCA protein detection kit. Different concentrations of protein standard and protein samples to be glued were added to the enzyme plate, and BCA was added to develop color.
4. After the quantification, determine the concentration of the protein according to the absorbance, and pack the protein separately. Add the same mass protein sample to the Buffer of 2%SDS protein lysis of the same volume, and add the buffer at 10:1. Make transient centrifugation and store them in the refrigerator at -80 ℃.
5. After preparing SDS polyacrylamide gel according to the instructions, install it into the electrophoresis tank, pour into the electrophoresis solution, add the same volume of protein sample to each pore, and add protein maker. Carry out electrophoresis for 40min under 80V constant voltage. After reaching the separation gel, increase the voltage to 120V constant voltage to continue electrophoresis for 1.5h. The running position of the band is determined according to the protein maker, and the electrophoresis was stopped at the right time.
6. After labeling the PVDF membrane, activate the PVDF membrane with methanol for a few minutes, and then immerse it in the transfer solution. According to the order of blackboard + filter cotton + filter paper + glue + PVDF membrane + filter paper + filter cotton + whiteboard, install the film under the condition of constant current 290mA and maximum voltage, about 90min.
7. After the end of the film transfer, remove the film and wash off the residual transfer solution with PBST. Then seal it with 5% milk (prepared with PBST) at room temperature for 1 hour.
8. After the closure, wash the remaining milk on the film with PBST, add an antibody (prepared with 5%BSA) and immerse it. Incubate it overnight at 4 ℃.
9. The next day, recycle the used first antibody. Wash off the residual first antibody with PBST every 10min and repeat it three times. Add the second antibody of the same species (prepared with 5% milk) and incubate at room temperature for 1 hour. Wash off the residual secondary antibody with PBST every 10min and replace it three times.
10. Develop on the strip developer and save the picture. The strips can be stored in the refrigerator at-20 ℃.
