Experiments

• Seamless Cloning
1. Vtotal=20μL.
2. Seamless cloning master mix:
1. ① EGF plasmid (after Restriction Endonuclease Enzyme Cut): 9.5mL
2. ② R plasmid: 0.5μL
3. ③ H2O: 0μL
4. ④ React 20 mins at 50℃.
3. Reserve in -20℃.
• Solid LB Medium
1. Add about 1.5%~2.0% agar to the liquid medium.
2. Heat it to 100 ℃ to dissolve, and pour it into culture dishes in a liquid state.
3. It will begin to solidify into a solid at about 40 ℃.
• Coating Plates
1. Before starting, hands and equipments should be disinfected.
2. Apply the bacterial solution and antibiotics (if necessary) evenly on the culture medium with a coating stick.
3. Seal it with tape and keep it in the incubator.
• Protein Gel Electrophoresis
1. Separating gel:
1. Assemble mold (the two glass plates should be aligned, with the longer glass plate 1.0mm).
2. Test for leaks with sterile water.
3. Add the ingredients in order of the ingredients list (from top to bottom) tenderly, add SDS to prevent bubbles from forming.



4. Put 4mL of the mixed liquid solution into the mold (to three fifths of the mold).
5. Seal with sterile water.
6. Wait for 40min-1h.
2. Stacking gel:
1. Pour water.
2. Add the ingredients in order of the ingredients list (from top to bottom) tenderly.



3. Wait for 10min-30min.
3. Electrolyzed water making:
1. Add following ingredients into a beaker.



2. Add 800mL water, stir until it dissolves.
3. Add water to 1L.
• Bacteria Collection
1. Pick 1mL from each bottle, in total 5 bottles.
2. 8000 rpm, 5min, 4 degrees Celsius. Contract once.
3. Pour out the supernatant, add 1mL again.
4. Repeat the last step 3 times, total 4 times. (*remain supernatant for once)
5. Use 800uL PBS resuspend, centrifugation
6. Repeat the last step, total twice.
7. Add 600 uL PBS resuspending.
8. Add 5X protein loading, liquid: loading=5:1
9. 95 degrees Celsius metal bath for 10 min.
10. Centrifugation, 12000 rpm, 15min.
• Expanding Cultivation
1. 5 bottles of 50mL LB, add 50μL antibiotics per bottle.
2. One kind of bacteria (EGF, R, EGF+R) 1mL, one bottle.
3. Shaker 37 degrees Celsius, 2h until OD600=0.5-0.6.
4. Add IPTG.
• Plasmid Extraction
1. Preparation:
2. Take 1.5-5mL of overnight cultured bacteria solution and use centrifuge for 2 minutes with revolving speed 8000xg , collect the thallus and abandon the medium.
1. ① Whether RNase A has been added to Buffer P1
2. ② Whether ethyl has even added to the Wash solution
3. ③ Whether precipitate has formed in Buffer P2 and P3
3. Add 250μL Buffer P1 to the precipitate and suspend the thallus throughly.
4. Add 250μL Buffer P2 and tenderly shake 5-10 times to mix the mixture fully after adding the solution and let it stand for 2-4 minutes.
5. Add 350μL Buffer P3 and tenderly shake 5-10 times to mix the mixture fully after adding the solution.
6. Centrifuge 5-10 minutes with revolving speed 12000xg. Shift in the supernate to the adsorption column, centrifuge 30 seconds with revolving speed 8000xg and dump the solution in the collecting pope.
7. Add 500μL Buffer DW1, centrifuge 30 seconds with removing speed 9000xg and dump the solution in the collecting pipe.
8. Add 500μL Wash Solution, centrifuge 30 seconds with revolving speed 9000xg and dump the solution in the collecting pipe.
9. Repeat 8 again to easy away ions in the centrifuge tube in order to keep the DNA pure.
10. Centrifuge the empty adsorption column for 1 minute with revolving speed 9000xg
11. Put the adsorption column in a clean 1.5mL centrifuge tube, add 50μL Elution Buffer on the centre of the adsorbed film without touching the film. Stand it in room temperature for one minute and centrifuge 1 minute. Then preserve the DNA solution in the tube.
• PCR Bacteria
1. Add 7μL of water, 1μL of forward primer, 1μL of bacteria, 10μL of enzyme.
2. Centrifuge the tubes for 10 seconds.
3. Put them into the PCR machine and pre-degenerate at 94°C for 2 minutes, degenerate at 94°C for 30 seconds, anneal at 60°C for 30 seconds, extend the DNA at 72°C for 30 seconds, terminal extend it at 72°C for 2 minutes. Run for 30 cycles in total.
• Protein fragmentation
1. Use ultrasonic instruments
2. 2.1/2 probe (power range: 60-600w), we use 35%,210w Run 4s, then stop5s
   EGF:25mins
   R:25mins
   EGF+R:±40min
• Electrophoresis
1. Measure 0.25g of agarose, 25 mL of buffer and use a microwave oven heat it until it turns colorless.
2. Cool it with water until it turns to about 50℃.
3. Add nucleic acid dye and pour it into a board to wool it often until it becomes a colloidal.
4. Put the colloidal to the electrophoresis machine and add buffer solution. Then load the samples.
5. Let the machine run for 20 minutes and put it under a gel imager and observe the image.
6. Gel recycle:
1. ① Use clean and sharp scalpel to cut DNA fragment from agarose gel. Measure and put into 1.5 mL centrifuge tube. (Ps: minimize gel block volume and let DNA remain in the gel.)
2. ② The concentration of agarose gel is 1% so add 300 μL Binding Buffer II into 100 μL agarose gel. Warm bath at 50-60 ℃ for 10min and intermittently shake and mix.
3. ③ Place the adsorption column into a 2ml collection tube. Transfer the mixture into adsorption column and let it stand for 2min. Centrifuge for 1 minute at 10000 rpm, dump and collect the liquid.
4. ④ Add 500 μL Wash Solution, centrifuge for 1min at 10000rpm. Dump and collect the liquid.
5. ⑤ Repeat step 4 once.
6. ⑥ Use spectrophotometer to measure the concentration of DNA we get is 173.5 ng/μL.
• Transformation
1. Place the competence DH5α, which is a clone vector that we can gain a large amount of plasmids from, on ice to melt.
2. After melting, 1 ml of plasmids are added and then place it for 30 minutes on the ice.
3. Put the competence to a 42℃ water bath for 90 seconds.
4. Place the bacteria on ice for 5 minutes.
5. Add 1 mL LB (non-resistance) and shake for 1 hour at 37℃.
6. Centrifuge 10 minutes with revolting speed 4000 rpm a room temperature.
7. Absorb the supernatant, remain 100-200 μL and resuspend the thallus.
8. Absorb the remaining bacterial fluid, and spread on the Amp board.
9. Place the board in 37℃ incubator for 12-16 hours.
• PCR
1. In an ice bath, add each component to a sterile 0.5ml centrifuge tube in the following order. There is a primer on 1 μL, and a primer on 1 μL, and a primer on 10 μL, Taq enzyme, 1 μL plasmid, 7 μL H2O.
2. Adjust your response program. The mixture was slightly centrifuged and immediately placed on a PCR instrument for amplification. Generally:
1. ① Pre- Duration(93 °C 4min 1 cycle)
2. ② Duration (94°C 30s)
3. ③ Anneal (45°C~ 68°C 30s 30cycle)
4. ④ Extend ( 72°C 1kb/min)
5. ⑤ Post- Extend ( 72°C 10min 1cycle)
3. After the reaction was finished, the PCR products were placed at 4℃ for electrophoresis detection or -20℃ for long-term storage.
• OPA
1. OPA solution preparation:



Add H2O (100ml).

2. Standard solution preparation:



Then taking 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL, of L- amino parodic acid to 25mL calorimetric tubes to make a constant volume of 25mL, and number them as 0,1,2,3,4 and 5 according to the concentration.

3. Determination of absorbance of standard solutions:

The specific method for measuring absorbance using the OPA method is as follows: first, adjust the wavelength of the enzyme-linked immunosorbent assay (ELISA) to 340nm, transfer 5 µL of water, 20 µL of the test solution, and 150 µL of OPA solution to a 96 well plate, and use a stopwatch to start timing. After 2 minutes, place it in the ELISA analyzer. Lastly, measure the reading and draw a standard curve.



(Standard curve: y=ax+b. y=absorbability, x=concentration)



0min - 5min = y

4. Determination method:
1. ① Solution preparations:

Weigh 1g of bovine serum protein and skimmed milk powder separately, dissolve them in water, transfer them to a 100mL volumetric flask, add water to volume, and prepare a 1% bovine serum protein solution and a 1% skimmed milk powder solution. Dilute them to the appropriate ratio as the mother solution, and dilute them to the same concentration as the test solution. (The protein concentration is approximate)

2. ② Determination of absorbance:

Add the prepared bovine serum albumin and EGF-R into the 96 well plate, adjust the wavelength to 340nm, and determine.(The absorbance at this time serves as a reference) Take 150 µ l of OPA solution and add it three times to the three wells in a 96 well plate. Add 5 µL (l2mg/ml) of protease K and 20 µL of two solutions (control group: equal volume PBS), and quickly place them in a micro-plate reader for reading. Set the power curve parameters to a total time of 30 minutes, with readings taken every 1 minute for the first 10 minutes, and readings taken every 5 minutes for the last 20 minutes. Then, put it into an enzyme reader for reading.

• Purified Protein
1. Buffer:



Add H2O until 23ml

2. M imidazole solution:



After fully dissolved, add H2O until 50mL

3. Imidazole buffer:



4. Purified Protein:
1. ① 100 mL of bacterial(E+R) liquid, centrifugal collection and precipitation.
2. ② Wash and precipitate twice with PBS buffer, then re-suspend. And crush though a LTHP cell crusher.
3. ③ 4 degrees centigrade, 400 rpm centrifuge 5 min, collect precipitation, clean twice with PBS.
4. ④ Re-suspension precipitate with binding buffer ,and then filter though 0.22 um filter membrane.
5. ⑤ -20 degrees centigrade for store.
1. Take out Ni column, let alcohol floe out. Wash the Ni column twice with ddH2O with 5 times the column volume.
2. Use the binding buffer balance column with 8 times the column volume.
3. Add the inclusion sample inside and mix well with the blue filter.
4. Put it on the ice and affinity on the horizontal shaker for 3-4h.
• Liquid LB Medium Preparation
1. Prepare 1% of tryptone, 0.5% of yeast, 1% of sodium chloride.
2. Mix the materials together and form a saffron liquid.
3. Use a sealing film and a kraft paper, and use a rubber band to seal the conical flask.
4. 4.Put the flasks into the high temperature sterilizer and sterilize it with the temperature of 121°C for 20 minutes.
• Preparation of receptor cells
1. The bacteria to be prepared into a sensitivity are frozen on the ice, and then laid on the antibiotic-free LB agar culture plate, 37°C for overnight cultivation.
2. Using aseptic operation, pick a bacterial colony from the agar plate, inoculate it into a culture solution of up to 500 mL, and culture it in a 37-degree shaker overnight.
3. During bacterial culture, a 0.1 mole concentration of calcium chloride solution and a 0.1 mole concentration of a calcium chloride solution containing 15% glycerin can be prepared and cooled after high-pressure sterilization.
4. Measure the absorption value to determine whether the bacteria have reached the logarithmic middle of growth, which is the stage where they are prone to DNA intake. Once the cells reach this stage, place them on the ice and keep them on the ice throughout the following process.
5. Divide the cells into two large centrifuge tubes, centrifuge them 4 degrees, discard the supernatant, and then re-suspend to the cooled 100mL 0.1 mole concentration of calcium chloride solution, and take an ice bath for 30 minutes.
6. After the last cleaning, use 50 mL of 0.1 mole concentration of calcium chloride solution containing 15% glycerol to reship the bacteria. Then, these bacteria are in a chemical sensitization.
• Enzyme cutting
1. Preparation of experimental materials
1. ① Material: plasmid DNA.
2. ② Reagents: restriction endonuclease, ddH2O.
3. ③ Instruments: micropipette, centrifuge, water bath pot, electrophoresis instrument, ultraviolet transmission observer.
2. Add to the 1.5 mL sterilization centrifuge tube in turn:
1. ① plasmid DNA: X μL.
2. ② Restrictive endonuclease: 1 μL.
3. ③ 10×buffer.
4. ④ ddH2O: make up 20 μL.
3. Mix well and mark it.
4. 37℃ water bath 1-3 h.
5. 70℃ water bath 10 min to stop the reaction.
6. Electrophoresis detects the effect of enzymatic cutting.
7. Results and analysis:

If an enzyme has only one enzyme cut site in the cyclic plasmid DNA, and the enzyme cut is thorough, and the electrophoresis results are detected under the ultraviolet lamp, the single enzyme cut should be one band, and the double enzyme cut should be two bands. If the number of bands is more than the theoretical value, it is possible that the enzyme is incomplete. If the result of enzyme cutting is the same as that of the plasmid band before the enzyme cut (super helix, linear and open loop), it means that the plasmid has not been cut at all.

• Hydrogel
1. Re-suspension the EGF-R inclusion body with urea solution, and dilute it to 10mg/mL and 5mg/mL protein solution after determining the concentration.
2. Dissolve 2g of sodium alginate in 100mL of ultra-pure water, and stir until it is completely dissolved. In the process, it can be appropriately heated.
3. Take 0.5g of CaCl2 and dissolve it in 10mL of ultra-pure water and prepare it into a 5% CaCl2 solution.
4. Take three 2mL EP tubes, add 0.5mL sodium alginate solution to each tube in turn, and then add 0.5mL of urea solution, 5mg/mL and 10mg/mL of protein solution into each tube separately, and fully blow and mix well.
5. Take 20µL of CaCl2 solution and add it to the three EP tubes in turn, and quickly blow and mix well.
6. Place it under 37℃ to form and dry for about 12 hours to take photos.
• Experimental design of EGF Cell Proliferation (CCK-8)
1. Bovine serum cells in good growth state are prepared into 5% cell suspension and added into 96-well cell culture plate, with 100uL (about 2×10³ /100uL cells) added into each well, and DMEM culture medium is added. There are four groups in total, one control group and three experimental groups.
2. Adding EGF, R and EGF-R protein separately into three experimental groups, make the final concentration be 20 ng/mL.
3. Incubate in a 37℃ incubator at constant temperature and humidity for 36h with 5% CO2 concentration.
4. Taking out appropriate amount of cells and photographed with Nikon TS100.
5. 10uL CCK-8 solution is added to 96-well cell culture plate and incubated in 37℃ incubator for 0.5-1 hours.
6. The absorbance is measured with a single wavelength of 450nm.
7. Formula to calculate the cell survival rate = [(As - Ab)/(Ac - Ab)] x 100% inhibition rate = [(Ac - As)/(Ac - Ab)] x 100% As: experimental hole absorbance (including cells, culture medium, CCK-8) solution and drug solution; Ac: control hole absorbance (including cells, media, CCK-8 solution, without drugs); Ab: blank hole absorbance (including culture medium, CCK-8 solution, excluding cells and drugs)