PART I PREPARATION

Culture Medium preparation

A. Apparatus

1. Electronic balance

2. Measuring Cylinder

3. Pure Water Equipment

4. Volumetric Flask

5. High-pressure sterilizer

B. Materials

1. LB Broth

2. ddH2O

C. Procedure

1. Wash the volumetric flask;

2. Add 25g LB Broth and 1L ddH2O;

3. Sterilization in the High-pressure sterilizer for one hour.

 

Agarose Gel Electrophoresis Preparation

A. Apparatus

1. Electronic balance

2. Erlenmeyer Flask

3. Microwave Oven

4. Measuring Cylinder

B. Materials

1. Agarose Powder

2. TAE Buffer

C. Procedure

1. Add 4.5g Agarose Powder into erlenmeyer flask;

2. Add 300ml TAE Buffer into the erlenmeyer flask;

3. Heat the solution in the microwave oven until it is clear;

4. Pour the liquid into the mold.

 

Cell resuscitation

A. Apparatus

1. Liquid Nitrogen Container

2. Centrifuge Tube

3. Centrifuge

4. Shaking Flask

5. Shaker

6. pipetting gun

B. Materials

1. Cell: HEK293F

2. OPM-293 Culture Medium

C. Procedure

1. Take the cell HEK293F out from liquid nitrogen container;

2. Put the cell into the centrifuge tube;

3. Add 10ml OPM-293 into the tube;

4. Centrifuge for 5min, 1000rmp;

5. Pour the supernatant fluid, and add another 25ml OPM-293;

6. Pour the mixture into the shaking flask;

7. Shake for 5 to 6 days.

 

PART II EXPERIMENT

Enzyme Digestion

A. Apparatus

1. Tube

2. pipetting gun

3. PCR machine

B. Materials

1. Heavy chain carrier model: PEE12.4-33D9H

2. Light chain carrier model: PEE6.4-33D9L

3. Enzyme Pfl 23 II

4. Enzyme Eco 72 I

5. Enzyme Cpol

6. ddH2O

7. Fast Digest Buffer

C. Procedure

1. Add the model of carriers into the tube, 5μL each;

2. Add Fast Digest Buffer, 5μL each;

3. Add Pfl 23 II 2.5 μL and Eco 72 I 2.5 μL into the tube containing PEE6.4-33D9L;

4. Add Cpol 2.5 μL and Eco 72 I 2.5 μL into the tube containing PEE12.4-32D9H;

5. Add ddH2O to the tube until the total volume reaches 50 μL;

6. Put the mixture into the PCR machine, 37 degree Celsius, 3 hours;

7. When reaches 3 hours, shift the temperature to 4 degree Celsius.

 

PCR

A. Apparatus

1. PCR machine

2. Tube

3. Pipetting gun

B. Materials

1. Model

2. Forward primer of light chain

3. Forward primer of heavy chain

4. Reverse primer of light chain

5. Reverse primer of heavy chain

6. PrimeSTAR MAX premix

7. ddH2O

C. Procedure

1. Add 25 μL PrimeSTAR MAX premix, 5 μL forward primer, 5 μL reverse primer, 1 μL model, and 14 μL ddH2O to the two system separately;

2. Set the procedure: 98°C 5 minutes, (98 °C 10 seconds, 54°C 15 seconds, 72 °C 45 seconds, 72°C 5 minutes)* 30 times, 4 °C for the remaining time.

 

Agarose Gel Electrophoresis

A. Apparatus

1. Gel bed

2. Electrophoresis tank

3. Comb

4. Pipetting gun

B. Materials

1. Gel red

2. DNA Marker

3. ddH2O

4. Electrophoresis buffer

5. Nucleic acid dye

C. Procedure

1. Add Dye: when agarose gel is cooled, add nucleic acid dye,and mix well;

2. Preparation of gel board: waterproof tape is pasted on both ends of the glue bed to form an 8mm high retaining wall, press the tape, and place it on the horizontal table. Insert the comb, and the lower end of the comb is about 1mm away from the bottom of the plate. Pour the gel into the glue bed continuously, 3-4mm high, avoid bubbles, and solidify at room temperature. After complete solidification, tear off the adhesive tape, place it in the electrophoresis tank, add the electrophoresis buffer, which is 1-2mm higher than the glue surface, gently pull out the comb obliquely from one end to remove the bubbles generated;

3. Prepare the solution: Add 1.8 μL plasmid, 2 μL gel red, and 16.2 μL ddH2O;

4. Sampling: add DNA maker first, then add DNA sample;

5. Electrophoresis: cover the electrophoresis tank, the sampling hole is located at the negative pole of the electric field, and connect the power supply, 140 V, 30 min. When the front edge of the dye moves to the bottom edge for 1-2mm, the electrophoresis ends;

6. Observation results: turn off the power, take out the gel bed, under the UV detector in the gel of plasmid DNA.

 

Gel Purification

A. Apparatus

1. Centrifugal tube

2. Centrifuge

3. Knife

4. Bain-marie

5. Column

6. Centrifuge tube

7. Stopwatch

B. Materials

1. Gel

2. Buffer GDP

3. Buffer BW2

4. Elution Buffer

C. Procedure

1. When the DNA fragment is separated, place the gel under ultraviolet light, quickly cut off the gel containing the target DNA fragment and try to remove the excess gel as much as possible;

2. Take the weight of the gel and transfer it to the centrifugal tube. Add 1 to 3 times the volume Buffer GDP. 50°C Water bath 10 to 15 minutes, let the gel block completely dissolve. During the bath, reverse the mixture of 3 accelerated lotions;

3. A short centrifuge collects droplets on the tube wall. Pack the HiPure DNA Mini Column in a 2ml centrifugal tube. Transfer≤700μl of the solution to the column. 12,000×g centrifugal intervals 30 to 60 seconds;

4. Drop the filter and put the columns back into the 2ml centrifugal tube. Transfer the remaining solution to the column.12,000 × g centrifugal intervals 30 to 60 seconds;

5. Drop the filter and put the columns back into the 2ml centrifugal tube. Add 300μl Buffer GDP to the column. Stand still for 1 minute. 12,000×g centrifugal intervals 30 to 60 seconds;

6. Drop the filter and put the columns back into the 2ml centrifugal tube. Add 600μl Buffer DW2;

7. Drop the filter and put the columns back into the 2ml centrifugal tube. 12,000×g centrifugal intervals 30 to 60 seconds;

8. Drop the filter and put the columns back into the 2ml centrifugal tube. Add 600μl Buffer DW2 to the column. 12,000×g centrifugal intervals 30 to 60 seconds;

9. Drop the filter and put the columns back into the 2ml centrifugal tube.12,000xg centrifuge for 2 minutes;

10. Add 15 to 30 μl Elution Buffer to the center of the column membrane in the 1.5ml centrifugal tube. Leave for 2 minutes. 12,000×g centrifuge for 1 minute. Drop the column and store the DNA at -20°C;

11. Short centrifuge, transferred to the sterilized 1.5 or 2ml centrifugal tube.

12. Add a Buffer GDP of equal volume, invert the tube;

13. Pack the HiPure DNA column in the collection tube. Transfer the mixture to the DNA column. 12,000×g off 30 to 60 seconds.

 

Chemical Transformation

A. Apparatus

1. Ice Box

2. Hot Tub

3. Shaker

B. Materials

1. Sensitive cells

2. LB culture medium

C. Procedure

1. Take 100 pL of the sensitive cells melted on the ice to add DNA gently mixed, on ice static 5min;

2. 42°C hot tub 45~60s, rapidly transferred to the ice bath static 2min. Add 700 microliters of LB liquid culture medium to the centrifugal tube;

3. Take the appropriate volume of recovery liquid uniformly applied to the antibiotic culture base containing amp, 37 ° C culture box reversed to cultivate overnight.

 

Incite the monoclonal culture

A. Apparatus

1. Pipet

2. Tube

B. Materials

1. Culture plate

2. LB Broth

3. Amp

C. Procedure

1. Take out the plate bacterial culture liquid;

2. Use a small pipette to place the monoclonal into the bacterial tube;

3. Add 2ml LB medium and Amp.

 

Plasmid Extraction

A. Apparatus

1. Centrifuge

2. Tube

3. Absorbent Paper

4. HiPure EF Maxi Column

B. Materials

1. Buffer E1

2. Buffer E2

3. Buffer E3

4. Buffer E4

C. Procedure

1. Pour the liquid to the 4 centrifugal tubes, 50 ml each;

2. Centrifuge, 4200rmp, 45min;

3. Pour out the supernatant, invert the tube and place it on absorbent paper to drain;

4. Add 2.5 ml Buffer E1 to each tube, mix the liquid;

5. Whirl the liquids for 10 seconds;

6.  Transfer the mixed bacterial liquid to one tube;

7. Add 10 ml Buffer E2, mix the liquids gently;

8. Add 10 ml Buffer E3 to neutralize, mix the liquid violently;

9. Centrifuge ;

10. Filtrate, remove the flocculent matter;

11. Add Buffer E4, mix;

12. Transfer half the liquid to the HiPure EF Maxi Column;

13. Centrifuge, 4200rmp, 3min;

14. Remove the filtrate;

15. Measure its concentration.

 

 

Transfection

A. Apparatus

1. Pipet

2. Tube

3. Super Clean Bench

B. Materials

1. Reduced serum culture medium CD05

2. Plasmid

3. PEI

C. Procedure

1. Prepare liquid A: 1ml CD05+20ug plasmid, rest for 5 min;

2. Prepare liquid B: 1ml CD05+60μl PEI, rest for 5 min;

3. Pour liquid B into A, rest for 20 min;

4. Pour liquid A and B into the cell HEK293F;

5. Shaker incubation at 37 degrees Celsius for 5-6 days.

 

 

Protein Purification

A. Apparatus

1. Pipet

2. Tube

B. Materials

1. Ultrasounded ddH2O

2. PBS phosphate Buffer

3. Tris HCl

4. Protein after transfection

C. Procedure

1. Prepare the ultrasounded pure water and PBS phosphate buffer;

2. Transmute the collected protein after 6 days;

3. Mix the three into the centrifuge 8,000 rpm centrifugal 30min;

4. Remove the cells to cleanse;

5. Use the ultrasounded pure water to wash Protein A for 3 times;

6. Wash the nickle column with PBS for 3 times;

7. Wash the protein with Tris HCl.

 

SDS-PAGE Analysis

A. Apparatus

1. Mini-PROTEAN Tetra cell

2. Comb

B. Materials

1. Resolver A

2. Resolver B

3. APS

4. TEMED

5. Stacker A

6. Stacker B

C. Procedure

1. Prepare Resolver solution: 2.5 ml Resolver A, 2.5 ml Resolver B, 25 μl 10% APS, 2.5 μl TEMED;

2. Fill the solution to 0.5 cm below the bottom of comb teeth;

3. Prepare Stacker solution: 1 ml Stacker A, 1 ml Stacker B, 10 μl 10% APS, 2 μlTEMED;

4. Fill the solution the the top of the model;

5. Insert the comb and allow polymerization for 30 min;

6. Run Gel.

 

ELISA

A. Apparatus

1. Enzyme calibration

2. Board-Washing machine

B. Materials

1. PBST

2. Anti-huma IgG FC-HRP

3. Casein

4. H2SO4

5. TMB

6. ADC

C. Procedure

1. Il13RA2-11His: 50 μl/well, 2 μg/ml;

2. 4°C, coated overnight;

3. Prepare 0.1% PBST cleaning solution;

4. Prepare 1% Casein closure solution;

5. Close for 1 hr, 37° C, 200μg/well;

6. Use 0.1%PBST to wash for three times;

7. Add ADC, 37° C, incubating for 1 hr, 50μl/well;

8. Use 0.1%PBST to wash 3 times;

9. Anti-human IgG FC-HRP 50μl/well. 37° C, incubating for 1 hr;

10. Use 0.1%PBST to wash 3 times;

11. Add TMB to show the color: 50μl/well, coloration for 10 min away from dark;

12. Add 50 μl/well 2M H2SO4 to end the coloration;

13. Detect the OD value at 450nm using enzyme calibration machine.