(1)Before use, add all RNaseA to Solution I and save it at 4 degrees.

(2)DNA Wash Buffer, was diluted with anhydrous ethanol as following the table and stored at room temperature.

(1)add 500 ul LB medium containing screening antibiotics to volumes of 2ml centrifuge tube , with target species 2-5ul, 37℃ oscillatory culture for 16 hours.

(2)After 16h, the bacterial fluid was relatively cloudy.If the lysis can not be performed immediately, it can be temporarily stored at 4 degrees.

(1)The bodies were collected at 20 ml and collected by centrifugation at 12000r/min at 10min, at room temperature.

(2)The medium was abandoned.Add 10 ml Solution I/RNaseA mixing liquid to the precipitation and mix with a pipette gun to completely resuspend the cells.

(3)Add 10 ml Solution II, and the centrifuge tube was gently reversed up and down 8 – 10 times to obtain clarified lysates.After reversal, the lysate was resting at room temperature for 2 – 3 min.

(4)The lid was covered with 5 ml precold N3 Buffer, and gently reverse up and down 10 times until a white flocculation precipitate was formed and quietly incubated for 2min at room temperature.

(5)Prepare a syringe filter, pull out the piston, place the syringe vertically on a test tube rack, and place a 50 ml centrifuge at the lower outlet of the syringe with the syringe opening upward.Immediately pour the lysate into the syringe of the filter. Cell lysates were maintained in the syringe for 5min.The white flocculation will float on the surface of the lysate.Cell lysates may have flowed from the filter syringe mouth.

(6)Cell lysates were collected using a new 50-ml tube.The syringe piston was inserted into the syringe and slowly pushed to flow the lysate into the 50 ml centrifuge tube.

(7)Add 1 x volume of ETR Solution (blue) to the outgoing filtered lysate and the tube was reversed 10 times and then resting in 10min in an ice bath.

(8)The above lysate was placed in a water bath at 42℃ for 5min.The lysate will appear cloudy again.At this time, centrifugation at 25℃, 4,000xg for 5min,ETR Solution would form a blue stratification at the bottom of the tube.

(9)The supernatant was moved to another new 50 ml tube, adding 5 x the volume of anaqueous ethanol at room temperature, and the tube was gently reversed 6-7 times for 1-2min. at room temperature.

(1)A HiBind DNAMaxi binding column was embedded into a 50 ml collection tube, and 20 ml superfiltrate was added to the HiBind DNAMaxi binding column.The cells were centrifuged at 4,000xg for 3min. at room temperatureAbandon filter.

(2)The HiBind DNAMaxi binding column was embedded into the same collection tube, and step 11 was repeated until all the remaining overfiltrate was bound to the HiBind DNAMaxi binding column and centrifuged under the same conditions.

(3)The HiBind DNAMaxi binding column was embedded into the same collection tube, added to 10 ml HBC Buffer to HiBind DNAMaxi binding column and centrifuged at 4,000xg for 3min, at room temperature.

(4)The HiBind DNAMaxi binding column was embedded into the same collection tube with 15 ml DNA Wash Buffer(diluted with anhydrous ethanol) to the HiBind DNAMaxi binding column and the filtrate was discarded by centrifugation at 4,000xg for 3min, at room temperature.

(5)The HiBind DNAMaxi binding column was embedded into the same collection tube with 10 ml DNA Wash Buffer(diluted with anhydrous ethanol) to the HiBind DNAMaxi binding column and the filtrate was discarded by centrifugation at 4,000xg for 3min, at room temperature.

(6)6000xg was emptied to dry the substrate of the HiBind DNAMaxi binding column for 10min.

(7)The HiBind DNAMaxi binding column was placed on a clean 50 ml centrifuge tube, adding 1 – 3 ml Endo-Free Elution Buffer to the HiBind DNAMaxi binding column matrix (added amount depending on the expected final product concentration) and resting for 5min at room temperature.

(8)It was centrifuged at 14,000xg for 5min to elute the DNA.

(9)The columns were discarded and the DNA product was stored at-20℃.

(1)Establish the following reactions on the ice. (Note: Add the enzyme to the reaction at last)

(2) Slowly blow and mix the reaction liquid and conducted instant centrifuge.

(3) Thermal insulation below 37°C for 5-15 minutes.

(4) Thermal inactivated in 20 minutes at 800 °C.

According to the electrophoresis diagram, the appropriate target fragment is mixed with the carrier (1:1-8:1 ratio of molecular weight) and connected by Infusion method. Connected at 37°C for 2 hours. If T4 joinase is used, stay overnight at 4°C.

(1) -80°CDH5α receptor cells melt on ice.

(2) 10μl bacterial liquid +0.5μl plasmid.

(3) Mix well and take an ice bath for 30 minutes.

(4) 42°C Heat for 90s.

(5) Ice bath for 5 minutes.

(6) Add 500μl nonresistant LB and put it into a 37°C rocker for 40 minutes.

(7) Centrifuge under 8000rpm for 5 minute.

(8) Inhale the upper clearance and leave 40μl heavy suspension.

(9) Coating board.

Select a single colony, lift it with the tip of a pipette, and mark it on a new LB plate.

(1)In step 6 of lysis of bacteria, inverted the centrifuge tube on clean absorbent paper allowed the removal of the media more thoroughly.

(2)In step 7 of the lysis of bacteria, to avoid intense mixing and lysates, the resting time should not exceed 5min, or otherwise break chromosome DNA and reduce the resulting plasmid purity.Exlonged resting time may lead to breakage of plasmid DNA.(When using SolutionII, cover the cap).

(3)The solution must be thoroughly mixed.If the mixture remains viscous and brown spherical, mixing needs to continue until full solution neutralization, which is essential to obtain a high yield.

(4)At steps 9 and 10 of the lysis of bacteria, a small fraction of the lysates may remain in the flocculation precipitation, do not reluctantly push the residual lysates into the filter, otherwise a small portion of the precipitation will flow into the collection tube.If any precipitation flowed into the overfiltrate, the precipitation was removed by centrifugation.

(5)Upon addition of ETR Solution, the lysates may appear turbidity, but will gradually turn to clarification after an ice bath.

(6) Concentrated DNAWashBuffer must be diluted in ethanol according to the instructions prior to use.If the DNA washing buffer is placed in the refrigerator before use, it must be taken out at room temperature.

(7) Enzymatic cutting can be done overnight.  

1. Remove the bacterial strain from the -80 ℃ refrigerator and immediately shake it in a 37 ℃ water bath until the liquid begins to melt and flow.

2. Prepare 1ml of DMEM culture medium and add it to a clean centrifuge tube. Also, add about 1ml of bacterial solution and dilute the DMSO cryopreservation solution.

3. Centrifuge (5 minutes, 800rpm), discard the supernatant, and resuspend the cells in 1ml of culture medium.

4. Take a culture dish (10cm) and add an appropriate amount of culture medium (about 6ml). Add the resuspended cells to it for propagation, and pass them once before use.  

1. Discard the old culture medium and add 1-2ml PBS to wash the cells. (optional).

After discarding PBS, add 6ml of new culture medium to the culture medium.

(Observing cell growth status and density under a microscope, generally 80% -90% passage is preferred)

1. Discard the old culture medium, add 1-2ml trypsin (cover the cells), and digest for 1 minute.

2. Add twice the volume of trypsin to the culture medium to terminate digestion, carefully blow it into a cell suspension, and transfer it into a centrifuge tube.

3. Centrifuge (800rpm, 5min) Add new culture medium to the new culture dish while centrifuging Large dish (10cm) 5-6ml Medium dish (65mm) 3-4ml Small dish (35mm) 1-2ml The thickness of the culture medium should be 2-3mm

4. Discard the supernatant, add 1ml of culture medium, and gently blow the cells with a pipette to resuspend them.

5. Load the cell suspension into 2-3 new culture dishes according to the culture plan and continue culturing in the incubator. (Generally, the same size of culture dish is passed from one to three, or the large dish is 400ul, the medium dish is 200ul, and the small dish is 100ul)

1. Lay the plate one day in advance, and when the cell density reaches 70% -80% under the microscope, transfection can be performed.

2. Dilute 5ng of plasmid with Opti-mem200 μ l and 15 μ l of lipoMax separately, mix well and leave at room temperature for 5 minutes.

3.After 3.5 minutes, mix the diluted plasmid with lipoMax and leave it at room temperature for 15-20 minutes to form a complex.

4. Drop the mixture into the cell culture dish and gently shake the dish to mix well.

5. Observe the transfection efficiency after culturing in the incubator for 36-48 hours.

When using it for the first time, prepare LAR II, which is the substrate of luciferase. Dissolve LAR II in LAR II buffer and store separately at -80 ℃ in the dark.

(1) Take a 24 well plate for cell microscopy and remove the cell chamber for subsequent operations;

(2) Take 100 µ l PBS and carefully add it to each well for cleaning. Vacuum pump suction, then add 100 µ l PBS along the hole wall;

(3) Add 100 µ l of luciferase reaction solution to each well, mix well with a shaking plate, blow and digest the cells, transfer to an empty EP tube and let it stand for 7-8 minutes. Measure the fluorescence value of luciferase on a Sirius instrument and try to complete the detection within 30 minutes;

(4) After measuring Luc, add 100 'µ l of Renilla reaction solution to each tube (1ml of Renilla detection solution mixed with 10ul of substrate, freshly prepared), mix well, let it stand for 7-8 minutes, and detect the fluorescence value of Renilla. The detection should be completed within 30 minutes as much as possible. After completion, export the data using a USB flash drive and close the software and instrument.

1.When the cell adhesion is not firm, try to digest at room temperature for 10-30 seconds, or reduce the concentration of trypsin to 0.05% for digestion.

2. Do not blow or beat cells when washing or digesting them with PBS or trypsin.