Protocol

Prepare LB+Amp plate

1. Prepare the medium by mixing:

   Component	Volume
   ddH2O	500ml
   Bacterial Agar	7.5g
   LB broth	12.5g
   Ampicillin (100 mg/ml)	100μl

2. Sterilize the solution (autoclave) before adding ampicillin, for 1 hour.
3. After sterilization, heat the molten agar mix by partially submerging the bottle in a 60°C water bath until it becomes fully liquid.
4. Add 100 μl of 100 mg/ml ampicillin to the medium and mix thoroughly by swirling.
5. Pour approximately 15 ml per plate, covering the surface evenly and avoiding bubbles.
6. Allow the plates to cool at room temperature with lids slightly ajar until solidified.

Prepare TB

Component	Volume
ddH2O	500ml
Glycerol	2ml
Terrific Broth	23.8g
Ampicillin	depend on the amount to aliquot (eg.Aliquoting 50ml TB (Amp 50 μg/ml), add 25 μl Amp 100mg/ml)

Prepare agar gel

Percentage	2%	1%
Agarose	2g	1g
1xTAE	100ml	100ml

1. Boil in a microwavable flask by microwave for 1-3 minute, until the agarose is completely dissolved.
2. Cool down the agarose solution for a while.
3. Add ethidium bromide (EtBr) to the agarose solution.
4. Pour the agarose into a gel tray with the well comb in place.

Pfu PCR

1. Prepare the PCR reaction mixture to a total volume of 20 μl.

   Component	Volume(μl)
   5X Phusion HF or GC buffer	4
   2.5 mM dNTPs	1.6
   10 µM Forward Primer	1
   10 µM Reverse Primer	1
   Template DNA	variable
   Phusion DNA Polymerase	0.2
   DMSO (optional)	0.6
   Nuclease-Free Water	to 20
   Total	20

2. Gently mix the reagents by pipetting up and down or flicking the tube.
3. Briefly centrifuge the tube to collect the liquid at the bottom.
4. Place the tube in a thermocycler and run the following routine PCR program:

Initial denature	98 ℃	3min	1 cycle
Denature	98 ℃	10s	25-35 cycles
Annealing	45-72℃	variable
Extension	72℃	15s/kb
Final Extension	72℃	5-10 min	1 cycle
Hold	4℃	∞

Gibson assembly

	2-3 fragments	4-6 fragments
recommend DNA ratio	Vector: Insert=1:2/1:3	Vector: Insert=1:1
Total amount of fragments	0.02~0.5 pmols Xμl	0.2~1 pmols Xμl
Gibson assembly Master Mix	10μl	10μl
ddH2O	10-Xμl	10-Xμl
Total	20μl	20μl

1. Incubate sample in a thermocycle at 50℃ for 50minutes when 2-3 fragments/60minutes when 4-6 fragments
2. Store on ice or -20℃ for subsequent transformation

Digestion

Component	Volume (μl)	Volume (μl)
Vector	2 μg	20 μg
10X buffer	2	5
restriction enzymes	0.2 + 0.2	0.2 + 0.2
ddH2O	to 20	to 50
Total	20	50

Ligation

1. Prepare the following components

   Component	Volume (μl)
   mole ratio (vector: insert) = 1:1	8
   T4 Ligase (NEB)	1
   10X Ligase buffer (NEB)	1
   Total	10

2. Set at room-temperature for 1~2hours or 16℃ overnight for ligation

Transformation

1. Thaw competent cells with room temperature or water bath for 10~20 seconds until 1/3 thaw competent cells with the ice bucket.
2. Mix ligation product and competent cells in the volume of 1:3.
3. Incubate on ice for 5 minutes.
4. Heat shock sample at 42℃ for 45 seconds
5. Cool down on ice for 1 minute.
6. Transfer to LB plate with desired antibiotics at 37℃, and spread transformed cells by rolling glass beads.
7. Immediately incubate the plate at 37℃ for 16~18 hours.

Colony PCR

1. Prepare 200μl tubes and 1.5ml tubes.
1. Add 9μl ddH2O into each 200μl tube
2. Add 80μl TB+Amp broth into each 1.5ml tube
2. Pick a single colony by tip into each tube, stir with 9μl ddH2O, and then stir with 80μl TB+Amp.
3. Prepare PCR mix for each reaction:

   Component	Volume (μl)
   GoTaq® Green Master Mix	10
   Primer(Forward)	0.5
   Primer(Reverse)	0.5
   Total	11

4. Add 11 μl PCR mix to each 200 μl tube containing the colony. Total reaction volume 20 μl.
5. Run PCR in a thermocycler using the following program:

   Initial denaturation	95 ℃	5min	1 cycle
   Denaturation	95 ℃	30s	30~40X Cycles
   Annealing	58℃	30s
   Extension	72℃	1kb/min
   Final extension	72℃	7 min	1 cycle
   Refrigeration	4℃	∞

Plasmid extraction

1. Harvesting
   • Transfer 1.5ml of cultured bacterial cells to a 1.5ml microcentrifuge tube
   • Centrifuge at 14-16000 x g for 1 minute at room temperature to form a cell pellet then discard the supernatant completely
   • Repeat the harvesting step as required for samples between 1.5-6.0ml using the same 1.5ml microcentrifuge tube
2. Resuspension
   • Add 200μl of PD1 Buffer (make sure RNase A was added) to the 1.5ml microcentrifuge tube containing the cell pellet
   • Resuspend the cell pellet completely by vortex or pipette until all traces of the cell pellet have been dissolved
3. Cell Lysis
   • Add 200 μl of PD2 Buffer to the resuspended sample then mix gently by inverting the tube 10 times
   • Let stand at room temperature for at least 2 minutes to ensure the lysate is homogeneous. Do not exceed 5 minutes
4. Neutralization
   • Add 200 μl of PD3 Buffer then gently mix immediately by inverting the tube 10 times.
   • Centrifuge at 14-16000 x g for 3 minutes at room temperature.
   • If using >5 ml of bacteria cells, centrifuge at 16-20000 x g for 5-8 minutes. During centrifugation, place a PDH Column in a 2 ml collection tube.
5. DNA Binding
   • Transfer all of supernatants to the PDH Column. Use a narrow pipette tip to ensure the supernatants is completely transferred without disrupting the white precipitate
   • Centrifuge at 14-16000 x g for 30 seconds at room temperature then discard the flow-through. Place the PDH Column back in the 2 ml Collection Tube.
6. Wash
   For Improved Downstream Sequencing Reactions
   • Add 400 μl of W1 Buffer into the PDH Column.
   • Centrifuge at 14-16000 x g for 30 seconds. Discard the flow-through then place the PDH Column back in the 2 ml Collection Tube. Proceed with Wash Buffer addition.
   For Standard Plasmid DNA Purification
   • Add 600μl of Wash Buffer (make sure absolute ethanol was added) into the PDH Column
   • Centrifuge at 14-16000 x g for 30 seconds at room temperature. Discard the flow-through then place the PDH Column back in the 2 ml Collection Tube.
   • Centrifuge at 14-16000 x g for 3 minutes at room temperature. Discard the flow-through then place the PDH Column back in the 2 ml Collection Tube.
7. Elution
   • Add 20μl elution buffer(preheat elution buffer at 55°C~60°C) and centrifuge at 14-16000 x g for 2minutes.
   • Save the elute.

Bacteria stock

1. Add 500 μl of the overnight culture to 500 μl of 50% glycerol in a 1.5mL tube (1:1). (Make the 50% glycerol solution by diluting 100% glycerol in ddH2O).
2. Freeze the glycerol stock tube at -80℃

Gel extraction

1. Excise the agarose gel slice containing the desired DNA fragment and trim away excess agarose to minimize the gel piece.
2. Transfer up to 300 mg of the gel slice into a 1.5 ml microcentrifuge tube and add 500 μl Gel/PCR Buffer.
3. Vortex briefly to mix, then incubate at 55–60°C for 10–15 minutes until the gel is completely dissolved, inverting the tube every 2–3 minutes during incubation.
4. If the solution turns purple, add 10 μl of 3 M Sodium Acetate (pH 5.0) and mix thoroughly, then allow the dissolved sample to cool to room temperature.
5. Place a DFH Column into a 2 ml Collection Tube and transfer up to 800 μl of the sample mixture into the column.
6. Centrifuge at 14,000–16,000 × g for 30 seconds, discard the flow-through, and return the column to the collection tube. Repeat loading if the sample volume exceeds 800 μl.
7. Add 400 μl W1 Buffer to the column, centrifuge at 14,000–16,000 × g for 30 seconds, discard the flow-through, and place the column back.
8. Add 600 μl Wash Buffer (with ethanol added), let it stand at room temperature for 1 minute, then centrifuge again at 14,000–16,000 × g for 30 seconds and discard the flow-through.
9. Centrifuge once more at 14,000–16,000 × g for 3 minutes to completely dry the membrane.
10. Transfer the dried DFH Column to a new 1.5 ml tube and add 20–50 μl of preheated (60–70°C) Elution Buffer or TE directly to the membrane.
11. Let it stand for at least 2 minutes, then centrifuge at 14,000–16,000 × g for 2 minutes to elute the purified DNA.

Bacterial protein induction

1. Preparation by cultured 5 ml bacteria in TB+AMP at 37℃ overnight.
2. Induction the protein expression in the bacteria by 1mM IPTG at 37℃ for 0, 2, 4, 6 hours. Then, quantify the OD600 value of induced bacteria. If OD600 reaches 0.2~0.4, save all bacteria as the induction sample.
3. Centrifuged non-induction and induction samples at 5000 rpm for 3minutes, and remove the supernatant.
4. Storage the pellet in -20℃ directly, or lyse the pellet with 100μl 1x sample buffer and store at -20℃.

Bacterial protein extraction

1. Centrifuging the sample at 5000 rpm for 3minutes if it didn’t centrifuge yet
2. Add lysis buffer(B-per/0.5%triton/8M urea)
   • B-per : Use Tube Revolver to rotate sample at room temperature for 15mins, and then centrifuge at 16000 x g for 20minutes.
   • Triton : Sonication 3x10" till the sample is no longer viscous, and then centrifuge at 13000 x g 4°C for 5minutes
   • Urea : Sonication 3x10" till the sample is no longer viscous, and then centrifuge at 13000 x g 4°C for 5minutes
3. Collect the soluble supernatant into a new tube.
4. Store soluble supernatant and the dried pellet at -20°C.

Protein quantification: BCA assembly

1. Do serial dilution of 10mg/ml stock of BSA diluting to 2mg/ml,1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml with PBS.
2. Prepare BCA Reagent mixture with BCA Reagent A and BCA Reagent B (A:B=50:1)
3. Add 10μl protein sample and standard BSA in 96 well plate.
4. Add 200μl BCA Reagent mixture to each well.
5. Store in 37℃ for 30 minutes.
6. Use Spectrometer to measure optical density in 562nm

Protein sample preparation

1. According to the result of BCA assembly to prepare sample(equal mass) in the tube.
2. Add sample buffer into tube.
   • Supernatant: According result to add sample and 4X sample buffer.
   • Pellet: Add 1X sample buffer.
3. Denature protein sample at 105°C for 5 minutes.
4. Cool down sample on ice for 10 minutes.

SDS-PAGE
Material:
Lower gel - for 2 gels

Percentage	14%	12%	10%	8%	6%
ddH2O	5.2ml	6.6ml	8ml	9.2ml	10.6ml
Lower gel buffer	5ml	5ml	5ml	5ml	5ml
30%(29:1)Acrylamide:Bisacrylamide	9.2ml	8ml	6ml	5.4ml	4ml
10%APS	0.2ml	0.2ml	0.2ml	0.2ml	0.2ml
TEMED	0.012ml	0.012ml	0.012ml	0.012ml	0.012ml
Total	19.812ml	19.812ml	19.812ml	19.812ml	19.812ml

Upper gel-for 2 gels

Percentage	6%	4%
ddH2O	6.6ml	7.4ml
Upper gel buffer	3ml	3ml
30%(29:1)Acrylamide:Bisacrylamide	2.4ml	1.6ml
10%APS	0.12ml	0.12ml
TEMED	0.012ml	0.012ml
Total	12.132ml	12.132ml

Fixing solution

Component	Volume
ddH2O	300ml
Acetic acid	50ml
Ethanol	150ml
Total	500ml

Staining solution

Component	Volume
Methanol	150ml
Acetic acid	50ml
Coomassie brilliant blue	1g
ddH2O	300ml
Total	500ml

Distaining solution

Component	Volume
ddH2O	300ml
Acetic acid	50ml
Methanol	150ml
Total	500ml

Experience protocol:

1. Wipe glass plates and spacers, then assemble them in a gel casting apparatus.
2. Mix the lower gel components for the resolving gel and pour into the gel plate.
3. Add 1 ml Isopropanol on top of the resolving gel to flatten the surface.
4. Wait for about 30 mins to solidified the resolving gel, and then remove the Isopropanol from the top.
5. Mix the components for the stacking gel and pour into the gel plate.
6. Insert the comb into the top of the spacers and wait for the gel to solidify.
7. Fill the running tank with 1× running buffer and place the cast gel into the tank.
8. Load 3 μl marker into the first well, then load appropriate protein samples into other wells.
9. Run the gel at 80 V until the dye front enters the resolving gel (≈20 minutes), then increase to 100 V until the dye front reaches the bottom of the gel (≈90 minutes).
10. Place the gel into gel-fixing solution for 10 minutes.
11. Stain the gel with staining solution for 10 minutes.
12. Distain the gel for 1.5–2 hours until the protein bands are clearly visible.

Ni-Sepharose Protein Purification Protocol

1. Replace of storage buffer of Ni-Sepharose with PBS
   • Take 100 μl slurry Ni-Sepharose beads, spin down at 600 rpm for 3 minutes, and discard the storage buffer.
   • Add 600 μl PBS (>with 10mM Imidazole, pH=7.4), and vortex to ensure complete resuspension of the beads.
   • Centrifuge for 1000 rpm for 3minutes to precipitate the beads, and discard the supernatant.
   • Repeat 3 times, then discard the PBS supernatant.
2. Binding
   • Add 400 μl lysate (overload) to the resin, rotate for 60 minutes, and centrifuge at 1000 rpm for 3 minutes to precipitate the beads.
   • Transfer the supernatant to a new 1.5 ml tube and save it as the flow-through sample.
3. Wash
   • Wash precipitated beads with 600 μl PBS (with 100 mM Imidazole, pH=7.4) by rotating for 10 minutes.
   • Centrifuge at 1000 rpm for 3 minutes to precipitate the beads and save the supernatant as washed samples.
   • The wash times depend on your experiment demand (suggest 2~3 times)
4. Elution
   • Eluted the binding protein from precipitated beads by adding 600 μl PBS (with 600mM Imidazole, pH=7.4), and rotating for 10 minutes.
   • Centrifuge at 1000 rpm for 3 minutes to precipitate the beads and save the supernatant for eluted samples

Cell Seeding and Passage

1. Observe the cells under a microscope to assess morphology and confluency.
2. Remove the spent medium, and wash the cells once with 3 ml PBS. Remove the PBS completely.
3. Add 3 ml trypsin to detach the ECM and dissociate the cells. Incubate the plate at 37 °C for 5 minutes.
4. Prepare a new culture plate by adding 4 ml pre-warmed DMEM.
5. Gently pipette the trypsin solution in the original plate to detach any remaining attached cells.
6. Transfer 1 ml of the trypsin–cell suspension into the new plate. Gently rock the plate back and forth and side to side to distribute cells evenly.
7. Return the plate to the incubator (37°C, 5% CO₂) and allow cells to attach.
8. After 24 hours, remove the medium containing trypsin.
9. Slowly add 5 ml of fresh pre-warmed culture medium along the sidewall of the plate to minimize cell disturbance.
10. Observe the cells under a microscope to confirm attachment and normal morphology, then return the plate to the incubator.

Plasmid DNA Transfection

1. The day before transfection, passage cells into a 6-well plate at ~1/6 of a confluent 10 cm plate. Cells will be 70–90% confluent at the time of transfection.
2. Use DMEM during seeding.
3. The day of transfection, remove DMEM from cells and replace with 1ml Opti-MEM.
4. For each well: Dilute 1.6 µg plasmid DNA in 250 µl Opti-MEM®. Mix gently.
5. In a separate tube, dilute 10 µl Lipofectamine 2000 in 250 µl Opti-MEM®. Mix gently and incubate for 5 minutes at room temperature. (For each cell)
6. Combine the diluted DNA and reagent (total 500 µl). Mix gently and incubate for 20 minutes at room temperature to allow complex formation.
7. Add 500 µl Opti-MEM® to each well. Remove the spent culture medium from the cells.
8. Add 500 µl DNA–lipid complexes dropwise to each well that containing 1 ml Opti-MEM®. Gently shake the plate back and forth to distribute evenly.
9. Incubate cells at 37 °C, 5% CO₂.
10. After 16 hours, replace the Opti-MEM® with 2 ml of fresh complete DMEM (with serum and antibiotics as required).

Western Blot

1. Run the SDS-PAGE.
2. Prepare electrophoresis tank soak with ice, and place the rotor and ice pack in the tank.
3. Fuel the tank with fresh or 1-time-recycled transfer buffer.
4. Activate the PVDF membrane with methanol for 5min. After that, change the methanol with transfer buffer (fresh one).
5. Prepare the other ingredients, including the sandwich, sponge, filter paper, and membrane, and soaking them in the transfer buffer.
6. After run the SDS-PAGE, taking the gel from the glasses and trimming it to remove the dye front and the useless parts.
7. Assemble the sandwich in the tanks, and under the surface of the transfer buffer and put it in the electrophoresis tanks.
8. Run electrophoresis in the cold room with constant 0.3A for 2 hours.
9. Open the sandwich and take out the PVDF membrane with tweezers, and trim the useless part of PVDF membrane on a glass cover with water or washing buffer.
10. Blocking the trimmed membrane with blocking buffer, then shaking it for 1hour at room temperature.
11. Removing the blocking buffer and wash it for 5, 5, 10, 10minutes with wash buffer (PBST, PBS with 0.1% Tween-20).
12. Soaking it with first antibody and shaking it in 4°C for overnight.
13. Next day. Wash the membrane for 5, 5, 10, 10min with wash buffer after recycle the first antibody.
14. Soaking it with secondary antibody and shaking it for 1hour at room temperature.
15. Remove the secondary antibody and wash the membrane for 5, 5, 10, 10minutes with wash buffer.
16. Apply ECL to the membrane and visualize the result

Immunocytochemistry (ICC)

1. Place coverslips into wells and coat with 0.1% gelatin (10 µl) or suitable coating. Incubate 10 minutes at 37 °C.
2. Seed cells at appropriate density and culture until desired confluency.
3. Wash cells 2 times with 500µl PBS. Fix with 500µl 4% PFA for 10–20 minutes at room temperature. Wash 3 times with PBS.
4. Permeabilize with 500µl (PBS+0.1% Triton X-100) for 5–10 min (if required). Wash 3 times with 500µl PBS.
5. Block the cells by incubating cells in the 50 µl mixture (FBS: PBS: DAPI=50:949:1), wait for 1hr.
6. Wash 3 times with 500µl PBS
7. Mount coverslips with 10µl mounting medium and seal edges.

Material:

Name	CAS number	Brand
Acetic acid, glacial	64-19-7	Macron Fine chemical
Chitosan	9012-76-4	EMPEROR CHEMICAL CO.,LTD.
Sodium hydroxide	1310-73-2	SHOWA CHEMICAL CO.,LTD.
Collagen I	9007-34-5	GEcoll Biomedical

Step:

1. Chitosan powder dissolves in 0.1M acetic acid at 50°C and stir overnight.
2. Mix Collagen solution and chitosan solution at a 1:1 (w:w) ratio and stir at 50 °C for 1 hour.
3. The mixed solution was placed at 4 °C overnight.
4. After removal from the refrigerator, transfer the solution to a Petri dish and add 0.1 M NaOH to adjust the pH to 6.
5. The solution was left to gel for 1 hour and placed at 4 °C overnight.
6. The gel was pre-frozen at −80 °C for at least 2 hours.
7. Samples were subsequently lyophilized.