Documented by Ding yunxian, Jiang Nan and Wu Litong

1. Recovering Plasmid DNA:

Materials:

• Overnight culture of bacteria transformed with plasmid
• PBS buffer
• Denaturing solution
• Renaturing solution
• 100% and 70% ethanol

Equipment:

• Eppendorf tube
• Desktop microcentrifuge
• Desktop vortexer

Procedures:

1. Grow an overnight culture of bacteria.
2. Centrifuge the culture to pellet the bacteria before proceeding with DNA preparation.
3. Remove the supernatant and resuspend the bacteria in buffer.
4. Add a denaturing solution from the DNA extraction kit to the resuspended bacteria.
5. Add a renaturing solution from the DNA extraction kit to the denatured bacteria.
6. Pellet the proteins and genomic DNA by centrifugation and remove the plasmid-containing supernatant.
7. Add ethanol (or isopropanol) to precipitate the plasmid DNA.
8. Centrifuge at 12000 rpm for 10 minutes to pellet the DNA.
9. Wash the pellet with 70% ethanol to remove excess salt.
10. Resuspend the DNA pellet with PBS buffer.

2. Diagnostic Endonuclease Digest

Materials:

• Plasmid DNA
• Restriction enzyme
• Restriction digest buffer
• Forward Primer
• Reverse Primer
• 5 μL 10X Taq buffer with MgCl2
• 1 μL dNTP mix
• Taq DNA Polymerase

Equipment:

• Thin-walled PCR tube
• Ice Bucket
• PCR Machine

Procedures:

1. In a 1.5mL tube combine the following:
1. 1 μg of plasmid DNA sample
2. 1 μL of each restriction enzyme
3. 3 μL restriction digest buffer
4. H₂O up to total volume of 30 μL
2. Mix gently by pipetting.
3. Incubate tube at 37 °C for 1 hour.
4. Place thin-walled PCR tubes on ice. Set up a 50 μL reaction:
1. 2 μL template DNA
2. 5 μL 10X Taq buffer with MgCl2
3. 1 μL dNTP mix
4. 2.5 μL of 10 μM Forward Primer
5. 2.5 μL of 10 μM Reverse Primer
6. 0.2 μL 5 units/μL Taq DNA Polymerase
7. H₂ up to total volume of 50 μL
5. Place reaction tubes in PCR machine. Set the standard time and temperature for each step in a PCR cycle:
1. Initial Denaturation for 2 minutes at 94°C.
2. Denature for 30 seconds at 94°C.
3. Anneal primers for 30 seconds at 5°C below Tm.*
4. Extend DNA for 2 minutes at 72°C.
5. Repeat b-d steps for 20-30 cycles.
6. Extension for 5 minutes at 72°C.
6. The products were stored at 4 °C.

3. Acquisition of Target Gene Fragment

Materials:

• PS Buffer
• template DNA
• Forward Primer
• Reverse Primer
• dNTP Mix
• Taq DNA Polymerase

Equipment:

• Eppendorf tube
• Desktop microcentrifuge
• Desktop vortexer
• Ice Bucket
• PCR Machine

Procedures:

1. Place thin-walled PCR tubes on ice. Set up a 50 μL reaction:
• 1 μL template DNA
• 10 μL PS Buffer
• 4 μL dNTP mix
• 1 μL of 10 μM Forward Primer
• 1 μL of 10 μM Reverse Primer
• 0.5 μL Taq DNA Polymerase
2. Place reaction tubes in PCR machine. Set the standard time and temperature for each step in a PCR cycle:
1. Initial Denaturation for 5 minutes at 98°C.
2. Denature for 10 seconds at 98°C.
3. Anneal primers for 30 seconds at 55°C below.
4. Extend DNA for 2 minutes at 72°C.
5. Repeat b-d steps for 20-30 cycles
6. Extension for 5 minutes at 72°C
3. The products were stored at 4 °C.

4. Construction of Recombinant Plasmids

Materials:

• Linearized vector plasmid DNA
• Target gene segment
• DNA ligase and buffer
• Competent state E. coli
• LB media

Equipment:

• Eppendorf tube
• Shaking incubator at 37 °C
• Stationary incubator at 37 °C
• Water bath at 42 °C
• Bucket of ice
• Microcentrifuge tubes
• Sterile spreading device

Procedures:

1. Combine the following in an Eppendorf tube:
1. 2.5 μL Plasmid Vector DNA
2. 2 μL Insert DNA (For a control group, add H₂O instead of insert DNA)
3. 2 μL Ligase Buffer
4. 1μL DNA Ligase
5. H₂O to a total of 10μL
2. Incubate at 37°C for 30 min before cooling in an ice water bath for 5 min.
3. Retrieve competent cells from -80°C refrigerator and defrost on ice for approximately 30 minutes.
4. Retrieve the agar plates containing the appropriate antibiotic from storage at 4°C, and let them warm up to room temperature. Leave in 37°C incubator for later use.
5. Mix 5μL of plasmid DNA into 50 μL of competent cells in a microcentrifuge tube. Gently mix the components. For a control group, add 1μL of water to verify that the LB agar’s antibiotic functions properly.
6. Incubate the competent cell/DNA mixture on ice for approximately 30 minutes.
7. Heat shocks each transformation tube by placing the bottom half of the tube into a 42°C water bath, leaving it for approximately 45 seconds.
8. Put the tubes back on ice for 2 minutes.
9. Add 250 μL LB media (without antibiotic) to the bacteria, and grow in 37°C shaking incubator for 45 min.
10. Plate an appropriate amount of the transformation onto the 10 cm LB agar plate containing the corresponding antibiotic. Incubate plates at 37°C overnight.

5. Agarose Gel Electrophoresis:

Materials

• DNA samples
• Gel loading dye
• Electrophoresis buffer
• TAE
• Agarose
• Gelred

Equipment:

• Well combs
• Gel box
• Microwave
• Electrophoresis chamber
• Voltage source
• UV light source
• Casting tray

Procedures:

1. Mix 1g of agarose powder and 8μL of GelRed with 100 mL 1xTAE in a microwavable flask.
2. Microwave for 1-3 min until the agarose is completely dissolved.
3. Let agarose solution cool down to about 50 °C in about 5 mins.
4. Pour the agarose into a gel tray with the well comb in place.
5. Place the gel at room temperature for 20-30 mins, until it has completely solidified.
6. Place the gel into the gel box of electrophoresis unit. Fill gel box with 1xTAE until the gel is covered.
7. Carefully load a molecular weight ladder.
8. Add loading buffer to each DNA sample. Carefully load samples into the additional wells of the gel.
9. Run the gel at 120 V for approximately 90 minutes.
10. Turn off power, disconnect the electrodes, and remove the gel from the gel box.
11. Visualize DNA fragments using UV light source.

6. Chondrocyte Transfection:

Materials:

• ATDC5 cells
• Firefly Luciferase plasmid
• Renilla Luciferase plasmid
• lipofectamine 2000
• Opti-MEM

Equipment:

• Cell incubator
• Cell culture plate
• Eppendorf tube
• Water bath
• Bucket of ice

Procedures:

1. ATDC5 cells were seeded in 24-well plates at 70% confluence one day in advance.
2. Combine the following in an Eppendorf tube A and incubated at room temperature for 5min:
1. 0.1 μg Firefly Luciferase plasmid
2. 0.02 μg Renilla Luciferase plasmid
3. 1 μL lipofectamine 2000
4. 100 nmol miRNA
5. Opti-MEM to 50 μL
3. The mixture was then incubated for 20 min at room temperature.
4. Combine the following in an Eppendorf tube B and incubated at room temperature for 5min:
1. 100 nmol miRNA
2. 1 μL lipofectamine 2000
3. Opti-MEM to 50 μL
5. Eppendorf tube A and B were mixed and incubated at room temperature for 20 min.
6. Each well was added with 200 μL of medium and 100 μL of transfection reagent. Transfection was performed at 37°C for 6 h, and the transfection medium was discarded and replaced with complete medium for another 48 hours.

7. Luciferase Activity Detection:

Materials:

• PBS
• Passive Lysis Buffer
• Luciferase Substrate
• Stop Reagent
• Renilla Substrate

Equipment:

• Eppendorf tube
• 96-well plate
• Microplate reader

Procedures:

1. Absorb the medium and wash twice with PBS.
2. Each well was added with 300 μL of Passive Lysis Buffer and shaken for 20 minutes at 4°C.
3. The lysate was aspirated into a 1.5ml centrifugal tube, centrifuged at 12000 rpm for 2 min at room temperature, and the supernatant was retained.
4. 20 μL supernatant was added to a black 96-well plate, followed by a rapid addition of 100 μL Luciferase Substrate. The firefly fluorescence intensity was detected immediately.
5. 100 μL pre-mixed Stop&Renilla Reagent was added to each well, the intensity of Renilla fluorescence was measured immediately.
6. Record the readings and calculate the ratio between the two sets of data.