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1. Preparation

1.1 Antibiotics

1.1.1 Ampicillin

Working concentration: 50 µg/mL

  1. Accurately weigh 0.5 g of ampicillin
  2. Measure 10 mL of water with a graduated cylinder
  3. Add ampicillin to the water and shake continuously until fully mixed
  4. Filter with a 0.22 µm filter to sterilize
  5. Dispense into small EP tubes

1.1.2 Carbenicillin

Working concentration: 50 µg/mL

  1. Accurately weigh 0.5 g of carbenicillin
  2. Measure 10 mL of water with a graduated cylinder
  3. Add carbenicillin to the water and shake continuously until fully mixed
  4. Filter with a 0.22 µm filter to sterilize
  5. Dispense into small EP tubes

1.1.3 Kanamycin

Working concentration: 50 µg/mL

  1. Accurately weigh 0.5 g of kanamycin
  2. Measure 10 mL of water with a graduated cylinder
  3. Add kanamycin to the water and shake continuously until fully mixed
  4. Filter with a 0.22 µm filter to sterilize
  5. Dispense into small EP tubes

1.1.4 Chloramphenicol

Working concentration: 25 µg/mL

  1. Accurately weigh 0.25 g of chloramphenicol
  2. Measure 10 mL of absolute ethanol
  3. Add chloramphenicol to the ethanol and shake continuously until fully mixed
  4. Dispense into small EP tubes

1.1.5 Erythromycin

Working concentration: 25 µg/mL

  1. Accurately weigh 0.25 g of erythromycin
  2. Measure 10 mL of absolute ethanol
  3. Add erythromycin to the ethanol and shake continuously until fully mixed
  4. Dispense into small EP tubes

1.2 Mediums

1.2.1 LB Medium

For a 1L medium preparation:

  1. Accurately weigh 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride
  2. Measure 1L of distilled water with a graduated cylinder
  3. Add the ingredients to a glass bottle and dissolve with ultrasonic waves
  4. Dispense into appropriate conical flasks
  5. Optional step: To prepare solid medium, add agar powder at a ratio of 15 g/L
  6. Seal the conical flask with sterile breathable sealing film
  7. Sterilize in an autoclave at 121°C for 20 minutes

1.2.2 MRS Medium

For a 1L medium preparation:

  1. Accurately weigh 52.3 g of MRS broth
  2. Measure 1L of distilled water with a graduated cylinder
  3. Add the ingredients to a glass bottle and dissolve with ultrasonic waves
  4. Dispense into appropriate conical flasks
  5. Optional step: To prepare solid medium, add agar powder at a ratio of 20 g/L
  6. Seal the conical flask with sterile breathable sealing film
  7. Sterilize in an autoclave at 121°C for 20 minutes

1.2.3 MRS Solution

For a 1L MRS Solution preparation:

  1. Accurately weigh 136.6 g of Sorbitol, 10 g of Glycine
  2. Measure 1L of MRS Medium with a graduated cylinder
  3. Add the ingredients to a glass bottle and dissolve with ultrasonic waves
  4. Dispense into appropriate conical flasks
  5. Seal the conical flask with sterile breathable sealing film
  6. Sterilize in an autoclave at 121°C for 20 minutes

1.2.4 SM buffer

For a 1 L medium SM buffer preparation:

  1. Accurately weigh 326 g sucrose, 0.71 g magnesium chloride
  2. Dilute with distilled water to a final volume of 1 L
  3. Mix the ingredients and shake until fully dissolved
  4. Sterilize in an autoclave at 121°C for 20 minutes

1.2.5 MRS-SM Recovery Solution

For a 100 mL recovery solution preparation:

  1. Accurately weigh 17.1 g of sucrose, 2.0 g of magnesium chloride
  2. Measure 100mL of MRS Medium with a graduated cylinder
  3. Mix the two and shake continuously until fully dissolved
  4. Filter sterilize using a 0.22 μm disposable flite

1.3 Other Solutions

1.3.1 1.0M IPTG Stock Solution

  1. Accurately weigh 2.383 g of IPTG
  2. Measure 10 mL of distilled water with a graduated cylinder
  3. Mix the two and shake continuously until fully dissolved
  4. Filter sterilize using a 0.22 μm disposable filter
  5. Dispense the solution into 1 mL aliquots and store at -20°C

1.3.2 10xPBS Buffer

For a 100 mL solution preparation:

  1. Accurately weigh 40 g of sodium chloride, 1 g of potassium chloride, 7.2 g of disodium phosphate, 1.2 g of potassium dihydrogen phosphate
  2. Measure 100 mL of distilled water with a graduated cylinder
  3. Mix the ingredients and shake until fully dissolved
  4. Sterilize in an autoclave at 121°C for 20 minutes

1.3.3 0.5 g/ml Sucrose

For a 100 mL solution preparation:

  1. Accurately weigh 50 g of sucrose
  2. Measure 100 mL of distilled water with a graduated cylinder
  3. Mix the ingredients and shake until fully dissolved
  4. Sterilize in an autoclave at 115°C for 20 minutes

1.3.4 0.1 g/ml Glycine

For a 100 mL solution preparation:

  1. Accurately weigh 10 g of glycine
  2. Measure 100 mL of distilled water with a graduated cylinder
  3. Mix the ingredients and shake until fully dissolved
  4. Sterilize in an autoclave at 121°C for 20 minutes

1.3.5 0.1 g/ml Magnesium Chloride

For a 100 mL solution preparation:

  1. Accurately weigh 10 g of magnesium chloride
  2. Measure 100 mL of distilled water with a graduated cylinder
  3. Mix the ingredients and shake until fully dissolved
  4. Sterilize in an autoclave at 121°C for 20 minutes

1.3.6 0.1 g/ml Calcium Chloride

For a 100 mL solution preparation:

  1. Accurately weigh 10 g of calcium chloride
  2. Measure 100 mL of distilled water with a graduated cylinder
  3. Mix the ingredients and shake until fully dissolved
  4. Sterilize in an autoclave at 121°C for 20 minutes

1.3.7 Transfer Buffer

For a 1L transfer buffer:

  1. Accurately weigh 68.46 g of sucrose and add to a glass bottle
  2. Measure 100 mL of glycerol and add to the bottle
  3. Dilute to 1L with pure water
  4. Shake the glass bottle to mix the solution
  5. Sterilize in an autoclave at 115°C for 20 minutes

1.3.8 Preparation of Agarose Gel

Materials: Agarose, 1x TAE, Graduated cylinder, Conical flask, Microwave, 1000x Golden View (nucleus dyes), Electronic scale

Steps:

  1. For a 1% agarose gel, mix the following in a conical flask:5 g of agarose powder, 50 mL of 1x TAE buffer
  2. Swirl the solution to mix well
  3. Heat the solution in the microwave until it boils
  4. Take out the conical flask and swirl until the solution is clear and without undissolved agarose powder
  5. Add 5 μL Golden View and swirl the solution
  6. Pour into the electrophoresis box, place the comb, and wait until it solidifies
  7. Remove the comb carefully

1.3.9 1x TAE Solution

Materials: 50xTAE, distilled water, graduated cylinder

Steps:

  1. Measure 100 mL of 50xTAE
  2. Measure 4900 mL of distilled water
  3. Mix both in a bucket and shake continuously until fully mixed

2. Molecular Biology Experiments

2.1 Preparation of E. coli Competent Cells for Chemical Transformation

  1. Streak glycerol bacteria onto a plate
  2. Pick a single colony and inoculate in 50 mL LB medium, incubate overnight
  3. Inoculate at 1% into 100 mL LB medium, culture to OD 0.6~0.7
  4. Pre-cool centrifuge, glycerol, 0.1 g/mL calcium chloride, and centrifuge tubes (50 mL, 1.5 mL)
  5. Centrifuge 100 mL bacterial culture in a 50 mL centrifuge tube at 4°C, 3600 rpm for 10 min
  6. Discard the supernatant, add 50 mL 0.1 g/mL calcium chloride, resuspend, and centrifuge at 3600 rpm for 10 min
  7. Discard the supernatant, add 50 mL 0.1 g/mL calcium chloride, resuspend, and place on ice for 30 min
  8. Centrifuge at 4°C, 3600 rpm for 10 min, discard supernatant
  9. Add 2 mL 0.1 g/mL calcium chloride and 1 mL 30% glycerol, resuspend and dispence into small tubes

2.2 Preparation of E. coli Competent Cells for Electroporation

  1. Inoculate E. coli into 5 mL LB medium, incubate overnight
  2. Add 50 μL of bacterial culture to 5 mL LB medium, culture to OD = 0.4~0.6
  3. Pre-cool bacterial culture and centrifuge
  4. Add 1 mL bacterial culture to a 1.5 mL sterile EP tube, centrifuge at 7000 rpm for 2 min, discard supernatant
  5. Add 1 mL pre-cooled sterile distilled water, resuspend, centrifuge at 7000 rpm for 2 min, discard supernatant
  6. Add 1 mL pre-cooled 10% glycerol, resuspend, centrifuge at 7000 rpm for 2 min, discard supernatant
  7. Repeat step 6
  8. Resuspend in 50 μL of 10% glycerol

2.3 E. coli Chemical Transformation

  1. Take ECN competent cells (do not touch the bottom with hands)
  2. Add 1 μL plasmid to 100 μL competent cells
  3. Place on ice for 30 min, heat shock at 42°C for 45 s
  4. Place on ice for 3 min, recover with 1 mL LB under a clean bench
  5. Incubate at 37°C in a shaker for 1 h
  6. Centrifuge at 5200 rpm for 2 min, resuspend bacteria, and plate

2.4 E. coli Electroporation

  1. Take ECN competent cells (do not touch the bottom with hands)
  2. Pre-cool electroporation cuvette
  3. Add 10 µL plasmid to 100 µL competent cells
  4. Add competent cells to the electroporation cuvette, place on ice for 5 min
  5. Perform electroporation at 2.5 kV
  6. Recover with 1 mL LB under a clean bench
  7. Incubate at 37°C in a shaker for 1 h
  8. Centrifuge at 5200 rpm for 2 min, resuspend bacteria, and plate

2.5 E. coli Protein Extraction

  1. Inoculate correctly transformed bacteria into 5 mL medium, incubate overnight
  2. Measure OD, inoculate into 50 mL 2×LB (initial OD=0.02), culture it for 24 h
  3. Measure final OD, centrifuge at 4°C, 3600 rpm for 10 min
  4. Take 90 µL supernatant + 10 µL loading buffer, place at 100°C for 5 min
  5. Resuspend bacterial pellet with pre-cooled 50 mL 1×PBS, centrifuge at 4°C, 3600 rpm for 10 min
  6. Discard supernatant, resuspend with pre-cooled 50 mL 1×PBS
  7. Break cells on ice, 3 s burst, 3 s pause, for a total of 15 min, probe inserted inside
  8. Take 90 µL of the lysate + 10 µL loading buffer, 100°C for 5 min
  9. Centrifuge at 3600 rpm, 4°C for 10 min
  10. Take 90 µL supernatant + 10 µL loading buffer, 100°C for 5 min
  11. Resuspend precipitate with 50 mL 1×PBS
  12. Take 90 µL supernatant + 10 µL loading buffer, 100°C for 5 min

2.6 Preparation of Lactiplantibacillus plantarum Competent Cells for Electroporation

  1. Inoculate 50 µL Lactiplantibacillus plantarum into 5 mL MRS medium, incubate at 37°C for 10-14 h
  2. Resuspend the bacterial suspension, inoculate 100 µl into 5 ml of MRS broth, and incubate statically at 37°C until OD600 = 0.6
  3. Centrifuge 5 mL of the culture at 4000 rpm for 10 minutes at 4°C using a refrigerated centrifuge. Resuspend the pellet in 800 µL of SM buffer and place on ice for 10 minutes.
  4. Centrifuge at 12,000 rpm for 1 minute at 4°C. Resuspend the pellet in 800 µL of SM buffer, place on ice for 10 minutes. Repeat this step three times.
  5. Centrifuging at 12,000 rpm for 1 minute at 4°C, add 90-100 µL of SM buffer to resuspend the pellet.
  6. Aliquot 50 µL per tube, store at -80°C

2.7 Lactiplantibacillus plantarum Electroporation

  1. Prepare electroporation cuvette, 2mL electroporation cuvette for Lactiplantibacillus plantarum, clean with absolute ethanol and air dry, expose to UV for 15 min on each side under a clean bench
  2. Preheat electroporator, take 50 µL competent cells, thaw on ice, add 5 µL plasmid, mix and add to electroporation cuvette
  3. Set electroporator to manual mode, voltage 2.5 kV, perform electroporation
  4. Immediately add 1 mL of MRS-SM recovery medium to the cuvette, mix and transfer to a centrifuge tube, incubate at 37°C for 2 h
  5. Spread the transformed cells onto an MRS agar plate and incubate overnight.

2.8 Plasmid Extraction

Materials: Centrifuge, pipette, P1, P2, P3, PW, BL, adsorption column CP3, collection tube

Steps:

  1. Column equilibration: Place adsorption column CP3 in the collection tube, add 500 µL BL, centrifuge at 12000 rpm for 1 min, discard waste liquid, and place adsorption column back into collection tube
  2. Take 1~5 mL overnight cultured bacterial culture, add to centrifuge tube, centrifuge at 12000 rpm for 1 min, and aspirate supernatant as much as possible (if bacterial culture is too much, multiple centrifugations can be performed to collect bacterial pellets in one centrifuge tube)
  3. Add 250 µL solution P1 to the centrifuge tube containing bacterial pellet, thoroughly resuspend bacterial pellet with pipette or vortex mixer
  4. Note: If bacterial clumps are not thoroughly mixed, it will affect lysis, resulting in low yield and purity
  5. Add 250 µL solution P2 to the centrifuge tube, gently invert 6-8 times to fully lyse bacteria
  6. Note: Gently mix, do not shake vigorously to avoid breaking genomic DNA, causing genomic DNA fragments to mix in the extracted plasmid. The bacterial culture should become clear and viscous, and the operation time should not exceed 5 min to avoid plasmid damage. If not clear, it may be due to too much bacterial culture, insufficient lysis, and bacterial amount should be reduced.
  7. Add 350 µL solution P3 to the centrifuge tube, immediately gently invert 6-8 times to mix well, white flocculent precipitate will appear, centrifuge at 12000 rpm for 10 min
  8. Note: After adding P3, mix immediately to avoid local precipitation. If there are still tiny white precipitates in the supernatant, centrifuge again and take the supernatant
  9. Transfer the supernatant collected in the previous step to adsorption column CP3 (place adsorption column in collection tube), try not to aspirate the precipitate, centrifuge at 12000 rpm for 30-60 sec, discard waste liquid, place adsorption column CP3 into collection tube
  10. Optional step: Add 500 µL protein removal solution PD to adsorption column CP3, centrifuge at 12000 rpm for 30-60 sec, discard waste liquid, place adsorption column CP3 back into collection tube
  11. If the host bacteria is an end A* host (TG1, BL21, HB101, JM series, ET12567, etc.), these hosts contain a large amount of nucleases, which easily degrade plasmid DNA, this step is recommended
  12. If the host bacteria is an end A* host (DH5α, TOP10, etc.), this step can be omitted
  13. Add 600 µL washing solution PW to adsorption column CP3, centrifuge at 12000 rpm for 30-60 sec, discard waste liquid, place adsorption column CP3 into collection tube
  14. Repeat step 8
  15. Place adsorption column CP3 in collection tube, centrifuge at 12000 rpm for 2 min to remove residual washing solution in adsorption column
  16. Note: Residual ethanol in washing solution will affect subsequent enzymatic reactions (enzyme digestion, PCR, etc.). To ensure that downstream experiments are not affected by residual ethanol, it is recommended to open the cap of adsorption column CP3 and place it at room temperature for a few minutes to completely dry the residual washing solution in adsorption material
  17. Place adsorption column CP3 in a clean centrifuge tube, add 50-100 µL ddH2O to the center of adsorption membrane, place at room temperature for 2 min, centrifuge at 12000 rpm for 2 min to collect plasmid solution into centrifuge tube

2.9 DNA Purification and Recovery

Materials: PC, BL, adsorption column CB2, collection tube, centrifuge

Steps:

  1. Place the gel with the band in an EP tube, add Buffer PC at a ratio of 300 µL per 50 mg gel, heat dissolve in a 60°C metal bath
  2. Place adsorption column CB2 in collection tube, add 500 µL BL, centrifuge at 12000 rpm for 1 min, discard waste liquid, place adsorption column back into collection tube
  3. Add the dissolved solution to adsorption column, place on ice for 5 min, centrifuge at 12000 rpm for 1 min, discard waste liquid, place adsorption column back into collection tube
  4. Add 600 µL washing solution PW, centrifuge at 12000 rpm for 30-60 sec, discard waste liquid, place adsorption column CB2 into collection tube
  5. Repeat step 4
  6. Place adsorption column CB2 in collection tube, centrifuge at 12000 rpm for 2 min to remove residual washing solution in adsorption column
  7. Air dry at room temperature for about 10 min to fully evaporate ethanol
  8. Place adsorption column CB2 in a clean collection tube, add 50-100 µL ddH2O to the center of adsorption membrane, place at room temperature for 2 min, centrifuge at 12000 rpm for 2 min to collect DNA into centrifuge tube

2.10 PCR

For a 15 µL system:

  1. Add 7.5 µL of 2×Taq enzyme, 0.8 µL of forward and reverse primers, 4.9 µL of water, and 1 µL of template to a PCR tube with a pipette
  2. Set annealing temperature and extension time in PCR machine according to primer and fragment length

2.11 Overlap PCR

  1. Bridging: Add 25 µL of Phanta Max Master Mix, x µL of Template 1, y µL of Template 2 and (25-x-y) µL of H₂O into the PCR tube. Place the tube in the thermocycler and start corresponding program
  2. PCR: Add 11 µL H₂O, 15 µL Phanta Max Master Mix, 1 µL of forward primer, 1 µL of reverse primer and 2 µL of the solution of Step1. Place the tube in the PCR amplifier and start corresponding program

2.12 Agarose Gel Electrophoresis

  1. Place the prepared gel in the electrophoresis tank
  2. Add 10 µL DNA Marker to wells
  3. Add 10 µL of the sample to be tested to wells
  4. Turn on electrophoresis equipment, run at 180V 250mA for 10 min

2.13 Seamless Cloning (Gibson Assembly)

  1. Design Primers: Design primers that will add overlapping sequences to the ends of DNA fragments. These overlaps should be complementary to each other and typically range from 20 to 40 base pairs
  2. Set Up the Gibson Assembly Reaction: Combine the following components in a PCR tube: 0.02–0.5 pmol of each DNA fragment with overlapping ends, Gibson Assembly Master Mix
  3. Incubate the Reaction: Incubate the reaction mixture at 50°C for 15–60 minutes. The time required may depend on the length and number of fragments being assembled
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