Team:CSU CHINA/Experiments
1.Molecular cloning process
1.1targeted gene gaining
1.1.1 PCR system
| Agentia | Dosage |
|---|---|
| Phusion (F549-L) | 0.5μl |
| HF Buffer (518) | 10μl |
| dNTP MiX (10X) | 1μl |
| Primer F | 1μl |
| Primer R | 1μl |
| DNA | 1μl |
| ddH20 | 35.5μl |
| Total | 50μl |
1.1.2 PCR process
| 98℃ | 3min |
|---|---|
| 98℃ | 30s |
| 58℃ | 30s |
| 72℃ | 1min |
| (2)-(4) repeat for 35 circulations | |
| 72℃ | 8min |
| 4℃ | —— |
1.2enzyme cutting
enzyme cutting system
(1)Establish the following reactions on the ice. (Note: Add the enzyme to the reaction at last)
| Component | 50μl reaction system |
|---|---|
| DNA | 1μg |
| 10XNEBuffer | 1X |
| Restriction Enzyme | 1μl |
| Nuclease-free Water | to 50μl |
(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.
Note: Enzymatic cutting can be done overnight.
1.3 connecting
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.
1.4 transform
(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 7000rpm for 1 minute.
(8) Inhale the upper clearance and leave 40μl heavy suspension.
(9) Coating board.
1.5 Pick a single coenobium to clone
Select a single coenobium, lift it with the tip, and underline the new resistance LB board.
1.6 coenobium PCR
1.6.1 PCR system
Compared with traditional therapy, there are many advantages in our design. For example, the system with 2 switches: blood glucose concentration and blue light exposure can realize the auto/semi-auto regulation, with higher security level. Feedback inhibition can prevent the hypoglycemia caused by the oversecretion of innsulin and improve the sensitivity of the system.
| Agentia | Dosage |
|---|---|
| Taq Mix | 5μl |
| dNTP Mix(10X) | 0.2μl |
| Primer F | 0.5μl |
| Primer R | 0.5μl |
| Bacteria | - |
| ddH20 | 3.8μl |
| Total | 10μl |
1.6.2 PCR process
| 94℃ | 3min |
|---|---|
| 94℃ | 30s |
| 58℃ | 30s |
| 72℃ | 1min |
| (2)-(4) repeat for 35 circulations | |
| 72℃ | 8min |
| 4℃ | —— |
1.7 sequencing
2.Transfection
BiYuntian Lipo8000 transfection reagent
2.1 Cell culture
(1)The cells were examined by mirror.Wet your hands with alcohol and remove cell dishes from the incubator; place them under a microscope for observation.Generation was performed when the cell density reached 70% – 80%.
(2)Turn off the UV light on the supernet workbench and turn on the white light and fan.Turn on the suction pump switch.
(3)Spray the dishes with alcohol and place them into a super-clean workbench.Spray the reagents to use (e. g., trypsin, bacon, PBS solution, etc.) and put alcohol into the supernet table.After spraying alcohol with both hands, you can operate in the super-clean workbench.
(4)Pull the bacon out with a suction pump.Draw 1 ml PBS solution to the media wall (avoid not directly beating the adherent cells to prevent damage) and shake the dish by hand so that the PBS solution is in full contact with the cells. PBS solution.
(5)One ml of trypsin digestion was absorbed and beaten directly to the bottom of the plate.After shaking, digestion was digested into a 37℃ incubator for three minutes and timing by timer.After three minutes, trypsin digestion was terminated by adding 1 ml culture medium to the plate.
(6)Prepare a 15-ml centrifuge tube.Bring the cells down the entire bottom from top to bottom (draw the liquid below with the gun and blow the cells down) and inhale into the 15 ml centrifuge tube.
(7)A further 15 ml centrifuge tube filled with 2 ml water was leveled with it, 1000rpm,3min.
(8)Two 10 cm dishes were prepared and 13 ml bacon was absorbed by electric gun and evenly divided into the two plates.
(9)After centrifugation, the supernatant was absorbed with a suction pump, leaving the cell precipitation (white).Add 1 ml bacon to blow, beat and mix.
(10)The 250ul(1:4) cell suspension was absorbed and was added evenly to the new dish (in the central circle, so that the falling suspension droplets form a circle for uniform distribution).Shake well and cross.In the horizontal direction, slide 10 times; the liquid stops shaking 10 times after the vertical direction.It can be repeated many times.
(11)After spraying alcohol, put in an incubator and "cross".
(12)After one day of culture, the cell density reached about 70-80% for transfection.
2.2 Transfection (with a six-well plate, for example)
(1)Six-well plates cultured with cells were replaced with 2 ml fresh medium (complete medium containing serum and antibiotics).
(2)The EP tube was taken, and if we needed to transfect cells from a six-well plate, (125 μl *6) D M E M medium was added to the EP tube and (2.5 μg *6) pl asmid DNA, was g ently blown with a gun.Add (4 μl *6) Lipo8000 transfection reagent, gently blowing with a gun and resting for 15-20min.
| 96-well | 48-well | 24-well | 12-well | 6-well | 6cm dish | 10cm dish | |
|---|---|---|---|---|---|---|---|
| No serum medium or Opti-MEM Medium was available | 5ul | 12.5ul | 25ul | 50ul | 125ul | 250ul | 750ul |
| DNA | 100ng | 250ng | 500ng | 1ug | 2.5ug | 5ug | 15ug |
| Lipo8000 transfection reagent | 0.16ul | 0.4ul | 0.8ul | 1.6ul | 4ul | 8ul | 24ul |
| Mix gently after adding DNA and Lipo8000 transfection reagent and then proceed directly to the next step without incubation at room temperature. | |||||||
| Amount of mixture added to each empty space | 5ul | 12.5ul | 25ul | 50ul | 125ul | 250ul | 750ul |
| A mixture of Lipo8000 transfection reagent and DNA was added evenly to each well at the above amount, and the culture was continued directly without subsequent replacement of the medium after hours. | |||||||
(3)The amount of 125 μl Lipo8000 transfection reagent-DNA mixture was added per well and evenly dropped to the entire well, followed by gently mixing.
(4)After continuing to culture for about 24 – 48 h, transfection effects can be detected in an appropriate manner, such as fluorescence detection, Western Blot, ELISA, reporter, etc.
2.3 Differences between the different transfection reagents
Bitian Lipo6000:
(1)The presence of antibiotics may be cytotoxic
(2)It is divided into A/B liquid matching, after each match, to be static for 5-20min.After the A, B liquid is mixed, stand still for 5min.
(3)The solution should be changed 4 – 6h after the transfection culture.(For Hela cells, media replacement was recommended 4 h after transfection, and for NIH3T3, CHO, HEK293T and HEK293FT cells 6 h after transfection)
(4)Formulations of A solution (containing DNA mixture) and B solution (mixture containing Lipo6000)
Lipo 2000:
(1)Is divided into A/B liquid match, after each match, to stand for 5min.After the A, B liquid was mixed, stand still for 15 – 20min.
(2)Hunger treatment was performed for 1h before transfection.The medium containing serum should be replaced with serum-free DMEM/Opti-MEM, for 1 h. in a 37℃ incubator
(3)Formulations of A solution (containing DNA mixture) and B solution (mixture containing Lipo6000)
(4)To be changed 4 – 6h after transfection culture, to replace with serum containing serum
1.For Lipo8000 transfection reagents, the presence of antibiotics does not affect transfection efficiency and does not cause cytotoxicity after cell transfection.
2.For transfection, DMEM medium (either high sugar DMEM or low sugar DMEM) or Opti-MEMMedium could be used
3.Pay special attention to non-Vortex or centrifugation when mixing DNA and Lipo8000 for centrifugation.After the preparation, the storage was stable for 6 hours at room temperature.
4.For cells with one well in the six-well plate, 30 μl dosage of Lipo8000TM transfection reagent can be appropriately adjusted in 2 – 6 μl, and DNA dosage is recommended to be fixed at 2.5 μg, but also in the range of 1 – 4 μg.Usually, the proportion of plasmid dosage (μg) and Lipo8000TM (l) is 1:1.6 or 1:2.5 is more commonly used. If necessary, the transfection effect can be optimized in the range of 1:0.5-1:5, and the recommended ratio in the above table is 1:1.6, when the amount of Lipo8000TM is relatively small and both economical and efficient.Optimal transfection conditions, depending on the cell types and culture conditions, can spontaneously optimize the transfection conditions within the range recommended above.
5.Plasmid concentration should be controlled within 0.5-5 μg/μl.Note 3: For multiple wells transfected with the same number of plasmids, the Lipo8000TM transfection reagent and DNA required for each well can be increased at the corresponding fold, and then mixed in the same centrifuge tube. Later, after mixing, droplets can be added to the cell culture vessel according to the recommended volume.Note 4: For other culture plates or culture vessels, the amount of various reagents can be converted in proportion to the culture area of the cell culture vessels.If transfection with oligonucleotides or RNA, etc., this could be performed with reference to the conditions for transfection with DNA.
3.Plasmid extraction
3.1 Preparation before the experiment
(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.
3.2 cells were cultured
(1)add 500 ul LB medium containing screening antibiotics to volumes of 2L conical flask , with target species 50ul,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.
3.3 lysed the bacteria
(1)The bodies were collected at 200 ml and collected by centrifugation at 4000xg 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
3.4 activated columns
(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℃.
Note:
(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.
4.enzyme linked immunosorbent assay
4.1 Introduction
Enzymelinked immunosorbent assay, ELISA, is an immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye.
4.2 Experimental method
The basic principle of ELISA is: 1. Bind antigens or antibodies to the surface of a solid-phase carrier and maintain its immune activity. 2 Connect the antigen or antibody to an enzyme to form an enzyme-marked antigen or antibody, which retains both its immune activity and enzyme activity. During the determination, the tested specimens (determin antibodies or antigens) and enzyme-marked antigens or antibodies react with antigens or antibodies on the surface of solid-phase carriers in different steps. The antigen antibody complex formed on the solid-phase carrier is separated from other substances by washing, and the amount of enzymes bound to the solid-phase carrier is proportional to the amount of substances tested in the specimen. After adding the enzyme-reacted substrate, the substrate is catalyzed by enzymes to become a colored product. The amount of the product is directly related to the amount of substances tested in the specimen, so it can be qualitatively or quantitatively analyzed according to the depth of the color reaction. Due to the high catalytic efficiency of enzymes, the reaction effect can be greatly amplified, so that the determination method can achieve a high sensitivity.
4.3 Experimental meterials
ELISA kit
ELISA plate、samples to be tested（cell suspension or supernatant or standard sample）、deionized water, etc.
4.4 Experimantal instruments
Enzyme-labeled instrument、shaker、pipette、EP tube
4.5 Experimental steps
4.5.1 Preparation ( Please read it before first experiment)
Open the kit, read the preservation condition of reagents
Melt the reagent under room temperature and balance the temperature
Melt the reagent under room temperature and balance the temperature
Add 25x Wash Buffer into deionized water to make it 1x concentration for plate washing
Shade the Color Reagent AB bottle with tin foil
There is insulin within human saliva, the pollution of exogenous insulin besides the sample should be prevented, wearing a mask is needed
4.5.2 Experiment
4.5.2.1 Prepare standard curves
Make standard insulin dissolution by adding deionized water into standard insulin, then perform gradient dilution
The 900μl calibration diluent RD5-62 is added to the left tube, and 500μl is added to the rest. 500μl is transferred 500μl for gradient dilution according to the method shown in the figure. Fully mix before each transfer to ensure accurate concentration. Calibrate the diluent as the 0 concentration detection point.
After dilution, the corresponding concentration standard sample is in each tube.
4.5.2.2 Enzyme-linked immune reaction
Balance the temperature of the required reagent. ( Don't take too long)
Remove the required slats from the frame, and put the rest back into the aluminum foil bag to seal it.
First, wash the board with Wash Buffer 300μl per hole twice. After the last washing, thoroughly absorb the liquid and pour it in a tissue for 3 minutes to dry completely.
A 150μl detection diluent RD1-105 is added per hole.
Add (standard/reference/sample) 50μl per hole. Cover it with the provided tape (can be replaced by plastic wrap). Incubate a room temperature vibration at 500±50rpm on a horizontal vibration bed for 2 hours.
Dry the liquid per hole, wash it repeatedly 4 times, and drain the liquid for the last time.
Each hole is added with 200μl Substrate Solution (made of an equal amount of Color Reagent AB, ready for ready-to-use, light avoidance), and room temperature lightproof hatching for 30 minutes. The color gradually changes from colorless to blue.
Add 50μl Stop Solution per hole. The liquid in the hole changes from blue to yellow. If there is green, it proves that the mixing is incomplete, tap and mix well.
4.5.2.3 Enzyme marker test
Test the enzyme marker within 30min
Turn on in the order of：Transformer -Enzyme marker-Computer
Open the operation software of the enzyme marker in the computer and set up the program
"Turn off Temperature Education-Moving Plate Medium 10s-Detection Wavelength 450nm".
Follow the prompts to place the enzyme marker in the designated position.
Read data and record.
Other settings remain unchanged, and the data at the corrected wavelength of 570nm is measured again.
Read data and record.
4.5.2.4 Draw standard curve/Calculate according to the standard curve
Subtract the corresponding data under 570nm from the data at 450nm to get the corrected accurate data.
The insulin concentration of the sample to be tested is obtained by using software to fit the standard curve/substit the data with the standard curve to convert the concentration.
4.5.3 Collation stage
Discard waste liquid, waste slats, adjust guns, and turn off the enzyme marker.
4.6 Cautions of this experiment
There is insulin within human saliva, masks should be worn before the experiment in case of exogenous insulin pollution.
The serum used for cultured cells also contains high concentrations of insulin, so relevant cell experiments (possibly including cultured cells) need to use insulin-free serum to avoid exogenous insulin contamination.
Insulin sample and insulin antibodies in the kit have high biological activity, inhaling and contacting should be prevented, use protections like gloves and masks
Stop Solution in the kit is acidic solution
The volatile of Color reagent Bin the kit is high, strong allergic reaction and strong upper respiratory tract stimuli might be caused by inhaling in. Inhaling should be prevented.
Washing steps during ELISA experiment are vital. Washing should be strictly conducted by steps to make sure the accuracy of the experiment. Otherwise, false positive and interference will be caused.
Switching tips during the experiment is important. This experiment is highly accurate, the ideal situation is change tips every sample adding.
The experimental results need optical path correction to eliminate the optical defects of the board. The specific method is to subtract 570nm from the data at 450nm. If the concentration conversion is directly with 450nm data, it may be high or low.
The seal of the aluminum foil bag that preserves the enzyme label plate must be sealed. There is desiccant in the bag. Keep the enzyme label plate dry during preservation, otherwise it may cause the inactivation of antigens or antibodies on the surface of the solid phase carrier.
5. Alginate hydrogel cultured cells
(1) Prepare 1% alginate solution, add ordinary medium (excluding serum double antibody) and 70 ℃ water bath (more than 30min is recommended) to completely dissolve sodium alginate powder (50ml system is recommended).
(2) Prepare cell suspension and adjust it to 10 ^ 7 / ml.
(3) Mix the cell suspension with sodium alginate solution in proportion, generally 4:1, and the final concentration is 10 ^ 6 / ml.
(4) adding alginate solution containing cells into the corresponding orifice (such as 24 hole plates), slowly (adding a circle along the wall, otherwise it will be very uneven) adding sodium alginate solution volume 1-2 times of crosslinking agent (5%CaCl2 solution) and crosslinking 10s to form a gel with certain structural strength, which can appropriately extend the crosslinking time as required.
(5) Add the medium with the same volume of sodium alginate solution for culture, and change the solution every 24 hours.