1.1. Luria-Bertani Broth preparation
1.1.1. Weigh 10g tryptone, 5g yeast extract, and 10g sodium chloride (2:1:2)
1.1.2. Mix the tryptone, yeast extract, and sodium chloride with double distilled water to 1L in total
1.1.3. Use the magnetic stirrer to mix the mixture thoroughly
1.1.4. Send the prepared LB broth to the autoclave for sterilization

1.2. Culturing the Bacteria
1.2.1. Add 250μl ampicillin solution into 25ml sterilized LB medium
1.2.2. Add 10μl Escherichia. coli solution into 7ml sterilized LB medium
1.2.3. Incubate the bacteria in LB medium in the shaker overnight (220rpm in 37 °C)

1.3. Plasmid Extraction
1.3.1. Dispense the overnight cultured bacteria into 2ml EP tubes, 1.7ml per tube.
1.3.2. Centrifugate of the bacterial solution (12000rpm for 2min at room temperature)
1.3.3. Remove the supernatant
1.3.4. Add 250μl Buffer SP1 and disperse the precipitated bacteria with a pipette
1.3.5. Add 250μl Buffer SP2 and mildly flip the EP tube 5-10 times then rest for 2-4min
1.3.6. Add 350μl Buffer SP3 and mildly flip the EP tube 5-10 times then centrifugate the solution (12000rpm for 10min)
1.3.7. Place the adsorption column into a new EP tube; then add 500μl Buffer S and centrifuge 12000rpm for 5min in order to balance the environment of the adsorption column
1.3.8. Transfer the supernatant from 1.3.6 into the adsorption column then centrifugate (12000rpm for 5min)
1.3.9. Remove the filtrate at the bottom of the EP tube
1.3.10. Add 500μl Buffer DW1 into the adsorption column then centrifugate (12000rpm for 1min) and repeat 1.3.9
1.3.11. Add 500μl wash solution (with anhydrous ethanol) into the adsorption column then centrifugate (12000rpm for 1min) and repeat 1.3.9
1.3.12. Repeat 1.3.11
1.3.13. Transfer the adsorption column into a new EP tube then centrifugate (12000rpm for 2min)
1.3.14. Add 50-100μl Elution Buffer on the adsorption membrane and rest for 1-2min then centrifugate (12000rpm for 2min)
1.3.15. Take 1μl product and measure the plasmid concentration by spectrophotometer

2.1.1. Mix 45ml Dulbecco’s Minimal Essential Medium (DMEM) and 5ml Fetal Bovine Serum (FBS) (9:1)
2.1.2. Take out the cells in the cryopreservation refrigerator and melt them in 37°C water
2.1.3. Add 500μl cells into the 5ml growth medium and dispense to 3 EP tubes
2.1.4. Centrifugate the cells (1000rpm for 3min at room temperature)
2.1.5. Remove the supernatant
2.1.6. Disperse the precipitated cells by pipette with 0.5ml growth medium
2.1.7. Pour 4ml growth medium into Petri dishes and inoculate cells
2.1.1. Mix 45ml Dulbecco’s Minimal Essential Medium (DMEM) and 5ml Fetal Bovine Serum (FBS) (9:1)
2.1.2. Take out the cells in the cryopreservation refrigerator and melt them in 37°C water
2.1.3. Add 500μl cells into the 5ml growth medium and dispense to 3 EP tubes
2.1.4. Centrifugate the cells (1000rpm for 3min at room temperature)
2.1.5. Remove the supernatant
2.1.6. Disperse the precipitated cells by pipette with 0.5ml growth medium
2.1.7. Pour 4ml growth medium into Petri dishes and inoculate cells

3.1. RNA Extraction
3.1.1. Remove the growth medium of the Petri dishes
3.1.2. Use 0.5ml Trizol and resuspend the cells and collect the cells into EP tubes
3.1.3. Rest the cells after lysed for 5mins under room temperature
3.1.4. Add 0.2ml chloroform into the EP tube and shake drastically then centrifugate (12000rpm for 10min)
3.1.5. Transfer 200μl upper aqueous phase (RNA) to new EP tube, then add 100μl anhydrous ethanol and blend completely
3.1.6. Put the adsorption column into the new EP tubes then add the solution from 3.1.5 on the adsorption membrane (12000rom for 30s)
3.1.7. Add 500μl RPE solution to the adsorption membrane and rest for 2min then centrifugate (12000rpm for 30s)
3.1.8. Repeat 3.1.7
3.1.9. Centrifugate the empty EP tube in 10000rpm for 2min
3.1.10. Add 30μl DEPC-treated ddH2O on the central of the adsorption membrane and rest for 5min then centrifugate (12000rpm for 2min)
3.1.11. Repeat 3.1.10

3.2. RNA Reverse Transcription
3.2.1. Add 1μl dNTPs Mix and 1μl Oligo-dT Primer to 2μl template RNA; then add RNase-free water up to 10μl
3.2.2. Put the template into the PCR amplifier with setting 65°C for 5min then rest at 4°C
3.2.3. Add 4μl 5× RTase Reaction Buffer, 0.5μl RNase Inhibitor, and 0.5μl Evo M-MLV-RTase into the 10μl annealed template then add the RNase-free water up to 30μl
3.2.4. Put the template into the PCR amplifier with setting 42°C for 15-30min then 95°C for 5min and rest at 4°C
3.2.5. Add 7.5μl TB green, 0.3μl Rox, 0.3 μl forward primer, 0.3 μl reverse primer and 4.6 μl ddH2O with 2μl cDNA template
3.2.6. Centrifugate the 96-well plate (1000rcf for 3min)
3.2.7. Proceed qPCR

4.1. Protein Extraction
4.1.1. Cell lysis Buffer was prepared according to 5X Buffer, 5× NaCl, 5× SDS, 5× DOC, and 5× NP-40.
4.1.2. Centrifuge at 700 ×g for 5 min and discard the supernatant
4.1.3. Wash the cells twice by resuspension in PBS then centrifuged at 4°C and remove the supernatant
4.1.4. Resuspended the precipitate in cell lysis buffer, immersed in an ice pack, and incubated on a shaker for 30min
4.1.5. Centrifuged the lysed cells at 12000 ×g for 10min at 4°C to precipitate cell debris
4.1.6. Transfer protein supernatant to EP tube and store at -80°C

4.2. SDS-PAGE Gel Preparation
4.2.1. Add the 3.7ml separation gel solution and separation gel buffer with 60μl modified coagulant
4.2.2. Pour a certain amount of solution into the plate to make the distance between the fluid level and the top edge of plate is 0.5cm longer than the comb
4.2.3. Pour ddH2O in the plate in order to compact the separation gel
4.2.4. Remove the water after the separation gel is solidified
4.2.5. Mix 0.75ml stacking gel solution and stacking gel buffer with 15μl modified coagulant
4.2.6. Pour the solution into the plate and then insert the comb
4.2.7. Leave it for about 15min for consolidation

4.3. Gel electrophoresis
4.3.1. Install the gel in the Electrophoresis and soak in the SDS-PAGE Running Buffer
4.3.2. Add 20μl protein template into the hole on the gel
4.3.3. Set the Electrophoresis for 90V

4.4. Western Blot
4.4.1. Add one pack of transfer solution and 200ml methanol then add ddH2O up to 1000ml
4.4.2. Cut the filter paper and membrane
4.4.3. Soak the filter paper, membrane, and gel in the transfer buffer
4.4.4. From the bottom to top: stent, sponge, 3 filter paper, gel, NC member, 3 filter paper, sponge, stent
4.4.5. Place the sandwich into the transfer room for 1h
4.4.6. Mix 5ml 20× TBS with 90ml ddH2O and 1 pack of blocking reagent to make the blocking solution
4.4.7. Soak the NC membrane into Ponceau S 10min
4.4.8. Soak the NC membrane into the blocking solution and place it on the shaker for 1h
4.4.9. Pour the blocking solution out and add new blocking solution into the box then add primary antibody of PDRG-1 to larger hole membrane, GAPDH to smaller hole membrane; culturing on the shaker in 4°C overnight
4.4.10. Add 10ml 20× PBST into 190ml ddH2O, then add 100μl Tween-80
4.4.11. Pour the primary antibody out, then add the mixture from 4.4.10 and shake for 3 times per 5min
4.4.12. Use TBS wash the NC membrane and soak it in the blocking solution then add the HRP secondary antibody and place it on the shaker for 1h
4.4.13. Pour out the blocking solution and wash the NC membrane by TBST on the shaker for 3 times per 5min; then wash it by TBS again
4.4.14. Prepare the DAB solution by adding 1 pack of DAB, 1ml 10× DAB development solution, 9ml ddH2O, and 10μl hydrogen peroxide
4.4.15. Place the NC membrane with adding developer in the Invitrogen E-Gel Imager