Materials

√         Buffer SP1 with RNase A: 250 μL

√         Buffer SP2: 250 μL

√         Buffer SP3: 350 μL

√         Buffer S: 500 μL

√         Buffer DW1: 500 μL

√         Wash Solution: 500 μL

√         Absorption column

√         Centrifuge

√         Collection tube

Procedure

1. preparation

Check whether RNase A has already been added into buffer SP1

Check whether ethanol has already been added to the wash solution

2. Adsorption column equilibrium

Add 500 μL Buffer S into the adsorption column, and centrifuge for 1min with 12,000 g.

Drain the waste liquid from the collection pipe.

Put the adsorption column back in the collection tube.

3. Collect bacteria for minutes and discard the medium. Take 1.5-5 mL of overnight cultured bacterial solution and centrifuge it by 8,000 g

4. Add 250 μL Buffer SP1 to the sediments and entirely suspend the bacteria.

Add 250 μL Buffer SP2 and gently reverse the centrifuge tube immediately

5. Centrifuge 5-10 times and mix well. Let the mixture stand at room temperature for 2-4 minutes.

6. Add 350 μL Buffer SP3 and gently reverse the centrifuge tube immediately

Mix 5-10 times.

7.12,000 g centrifuge for 5-10 min. Transfer the supernatant into the adsorption column,

Centrifuge the supernatant at 8,000 g for 30 seconds and drain the liquid in the collection tube.

8.(Optional) Add 500 μL Buffer DW1 and centrifuge 9,000 g, 30 s. Drain the collection tube.

9. Add 500 μL Wash Solution and centrifuge 30 s by 9,000 g

10. Repeat Step 9.

11. Centrifuge the empty adsorption column at 9,000 g for 1 min. Drain the ethanol from the adsorption column.

12. Place the adsorption column into a clean 1.5 mL, add 50-100 μL Elution Buffer, place it at room temperature for 1 minute, and then centrifuge it for 1 minute.

Materials

√         5’ primer: 4 μL

√         3’ primer: 4 μL

√         PCR mix: 50 μL

√         ddH2O: 42 μL

√         PCR tube

Procedure

1. Put all reagents on ice

2. Mixing 50 μL primers (3’primer 4 μL, 5’primer 4 μL, plasmid 2 μL, water 42 μL) into a homogenous state and transfer the solution into the PCR tubes

3. Placing tubes in the PCR machine

Prepare negative control without template DNA

Prepare positive control with template DNA of known size and appropriate primers

conditions: at 95℃double helical DNA unwinding, at 65℃ primer matching with DNA template, at 72℃ DNA replication

Materials

√         TAE:120ml

√         Agarose:1.20 g

√         Nucleic acid dye:5μL

√         Machine

√         Mode

Procedure

√         Preparing TAE (TAE powder and distilled water)

√         After adding 120 ml TAE and 1.2g Agarose into the flask, heat the solution in the microwave.

√         Waiting for 3 to 5 minutes to cool down the solution naturally.

√         Adding 5 μL nucleic acid dye in the solution.

√         Adding the solution into the mode.

Materials

√         Wash Solution added with ethanol

√         Buffer B2

√         Water bath

         Wash solution: 500 μL

         Elution Buffer:15-40 μL

√         1.5 mL centrifuge tubes

Procedure

1. Turn the water bath to 50℃.

2. Cut the agarose gel containing the target DNA fragment and weigh it out.

3. Add Buffer B2 3-6 times the weight of the gel, 50°C water bath for 5-10 minutes.

4.(Optional) For <500 bp segments, add isopropyl alcohol that is 1/3 Volume of Buffer B2.

5. Transfer the solution into the adsorption column and centrifuge for 30 s at 8,000 g.

6. Add 500 μL Wash Solution and centrifuge 9,000 g for 30 seconds.

7. Repeat Step 6.

8. Centrifuge the empty adsorption column at 9,000 g for 1 min.

9. Put the adsorption column into a clean 1.5 mL centrifuge tube and drain the ethanol from the collection column.

10. Add 15-40 μL Elution Buffer to the center of the collection column, let it stand at room temperature for 1 minute, and centrifuge for 1 minute. Then, preserve the DNA solution in the tube.

Materials

√         pET 24a: 4 μL

√         DNA fragment: 1 μL (according to the length)

√         2* Clon Express reaction system: 5 μL

Procedure

√         Adding all materials into tubes

√         Water bath at 50 ℃ for 15 minutes

Materials

√         homologous recombination system products

√         receptive cells: 100 μL

Procedure

1. The 10 μL homologous recombination system was extracted and added to 100μL receptive cells.

2. First ice bath for 30 minutes

3. Heat shock 42℃.

4. Then ice bath for 2 minutes, add 900 μL liquid LB.

5. Culture in Petri dishes.

The construction of an expression vector for collagens a, b, c, d, and e was undertaken to facilitate the production of fusion proteins in E. coli BL21 (DE3). The plasmid DNA was successfully transformed into E. coli BL21 (DE3), enabling the expression of the collagens above using an auto-induction technique. The induction of collagens a, b, c, d, and e was carried out using the auto-induction method in E. coli BL21 (DE3) at a temperature of 18 °C. Following a 36-hour induction period, the cells were harvested via centrifugation. The resulting cell pellet was resuspended in a lysis buffer comprising pH 8.0, 25 mM Tris-HCl, 300 mM NaCl, 0.5 mM TCEP, 1 mM PMSF, 0.1 mg/mL lysozyme, and 25 U/mL SuperNuclease, and incubated for 30 minutes at room temperature. Subsequently, the cells were lysed by sonication on ice, and the cell debris was removed by centrifugation at 18,000 rpm at 4 °C for 30 minutes. The supernatant was then collected and subjected to purification using Ni-TED agarose. Further collagens a, b, c, d, and e purification was achieved on a Superdex 75 10/300 GL column with a protein storage buffer consisting of pH 7.4, 25 mM Tris-HCl, 300 mM NaCl, and 0.5 mM TCEP.

Materials

√         SDS-PAGE gel mold set

√         SDS-PAGE color preparation kit

√         Coagulant TEMED

√         Electrophoresis buffer

Procedure

1. Assemble the glue mold and configure the lower layer of glue first (take a piece of 0.75/1.00/1.50mm thick mini glue as an example).

2. Take 2.0/2.7/4 mL of the lower-layer glue solution (2X) and the lower-layer glue buffer (2X) in the same volume and mix it well.

3. Add the improved coagulant 40/55/80 μL to the mixed solution in Step 2 and stir gently to avoid bubbles.

4. Add the lower layer of glue solution mixed in step 3 to the glue-making mold so that the liquid level is 1.5 cm away from the upper side of the glass plate, and then cover the lower layer of glue solution with a layer of water or alcohol to keep the gel surface flat and do not produce bubbles.

5. Stand at room temperature (25°C) for 6 to 10 minutes, and when the boundary between the lower layer and the covering phase appears, the gelatin is said to have solidified.

6. Configure the upper layer glue.

7. Slowly pour off the covering phase, take the same volume of the upper adhesive solution (2X) and the color upper adhesive buffer (2X), 0.5/0.75/1.0 mL each, and mix them in a new dispensing cup.

8. Add the modified coagulant 10/15/20 μL to the mixed solution in step 7 and stir gently to avoid bubbles.

9. Add the upper glue solution to the upper layer of the lower glue until the gel solution reaches the top of the glass plate. Slowly insert the comb into the gel to avoid bubbles.

10. Stand for 10~15 minutes, wait for the upper layer of glue to solidify, carefully pull out the comb, use the gun head or syringe to absorb the electrophoresis buffer, and wash the sample hole clean then the SDS-PAGE electrophoresis operation can be performed.

Take 250 mL of methanol solution, add 0.5 g of Coomassie bright blue R250 powder, stir the solution thoroughly, add 50 mL of acetic acid, mix again, and finally add 500 mL of distilled water. Store the dye solution at room temperature.

70 mL of glacial acetic acid, 50 mL of anhydrous ethanol, and distilled water to 1 L.

Store at room temperature.

A 10% native-PAGE gel for electrophoresis was prepared by combining 2.5 mL of 40% acrylamide solution, 2.5 mL of 1.5 M Tris-HCl buffer at pH 8.8, and 5 mL of deionised water. The polymerisation was initiated by adding 50 μL of 10% ammonium persulfate and 10 μL of TEMED. For sample preparation, 20 μL of a 0.5 mg/mL collagens a, b, and c-d-e (1:1:1) solution were mixed with 5 μL of sample buffer containing 250 mM Tris-HCl at pH 6.8, 50% glycerol, 0.5% bromophenol blue, and 0.05% Triton X-100. The samples were loaded onto the gel and electrophoresed in a Tris-glycine running buffer of 25 mM Tris-HCl, 250 mM glycine, and 0.01% Triton X-100. After electrophoresis, the gel was stained with Coomassie Brilliant Blue and destained using a solution with 10% methanol and 7% acetic acid for enhanced clarity.

SEC was performed with a SuperdexTM 75 Increase 10/300 GL analytical column (GE Healthcare) at 4 ℃. The column was pre-equilibrated and eluted with buffer (25 mM Tris-HCl, 300 mM NaCl, 0.5 mM TCEP, and 0.01% Triton X-100, pH 7.4) at a 0.3 mL/min flow rate. Approximate 0.5 mg/mL collagens a, b, and c-d-e (1:1:1) were pre-incubated separately in 0.5 mL volume for 63 min at 37 ℃ before loading onto the column at a 0.3 mL/min flow rate.