LB Liquid Medium (100 mL)

  1. Prepare the following mixture in an Erlenmeyer flask.
    Element Volume or Weight
    NaCl 1g
    Peptone 1g
    Yeast 0.5g
    Water 100mL
  2. After they fully dissolve, transfer the liquid to a 250mL Erlenmeyer flask and seal it.
  3. Sterilize in a high-pressure steam sterilizer.

LB Solid Medium (100 mL)

  1. Prepare the following mixture in an Erlenmeyer flask.
    Element Volume or Weight
    NaCl 1g
    Peptone 1g
    Yeast 0.5g
    Agar Powder 1.5g
    Water 100mL
  2. After they fully dissolve, transfer the liquid to a 250mL Erlenmeyer flask and seal it.
  3. Sterilize in a high-pressure steam sterilizer.

Agarose Gel Electrophoresis (30 mL, 1% Pore Gel)

  1. Prepare a small gel plate and a small comb.
  2. Weigh 0.3g of agarose.
  3. Add 30mL of 1× TAE.
  4. Heat in a microwave oven until the agarose is completely dissolved.
  5. When the solution cools to below 60°C, add 3μL of GelGreen nucleic acid staining dye and mix thoroughly.
  6. Pour the solution onto the flat plate with the comb inserted and use a pipette to remove any bubbles.
  7. After solidification (about 30 minutes), carefully remove the comb and soak the gel in 1× TAE buffer.
  8. Mix the sample with 6× DNA loading buffer at a volume ratio of 5:1. Add the appropriate amount of DNA marker and sample to the sample wells.
  9. Open the power supply, set the voltage to 100V, and start running until the bromophenol blue dye reaches the bottom of the gel.
  10. Remove the gel from the electrophoresis instrument and observe it under a fluorescence projector.

Polyacrylamide Gel Electrophoresis

  1. Assemble the gel-making device and perform a leakage test (add water to observe any leaks).
  2. Prepare the separation gel using the following concentrations:
    Gel Concentration Water (mL) 30% Acr-Bis (mL) Separation Gel Buffer (mL) Modified Ammonium Persulfate (μL)
    10% 2.08 1.67 1.25 20
    12% 1.75 2.0 1.25 20
  3. Note: Add modified ammonium persulfate at the end and shake it immediately before pouring it into the glass plate.
  4. Seal the liquid surface with water or isopropanol.
  5. Prepare the concentrated gel using the following volumes:
    Gel Volume Water (mL) 30% Acr-Bis (mL) Separation Gel Buffer (mL) Modified Ammonium Persulfate (μL)
    2mL 1.14 0.34 0.5 10
  6. Note: Add modified ammonium persulfate at the end and shake it immediately before pouring it into the glass plate.
  7. Insert the comb and wait for the gel to solidify completely.
  8. Prepare samples:
    1. Preparation of bacterial samples:
      • Centrifuge 200μL of bacterial culture for a certain time at 12,000 rpm for 3 minutes, discard the supernatant, resuspend the pellet with 20μL of sterile water, and then mix with 5μL of 5× SDS loading buffer.
      • Boil the sample in boiling water for 8 minutes, immediately ice-bath for 3 minutes, and then centrifuge at 12,000 rpm for three minutes. Take the supernatant when loading.
    2. Preparation of supernatant samples after cell disruption:
      • Fully mix 20μL of supernatant with 5μL of 5× SDS loading buffer.
      • Heat the sample at 98°C for 12 minutes in a PCR instrument, and load the samples after cooling.
  9. After gel solidification, carefully remove the comb, place the plate into the electrophoresis tank, add 1× SDS buffer to the electrophoresis tank to fill it, and then carefully add the sample to the sample wells.
  10. Open the power supply, set the voltage to 100V, and change to 120V when the dye enters the separation gel. Stop electrophoresis when the dye moves to 1cm from the bottom.
  11. Remove the gel, cut off the corners, dye with Coomassie brilliant blue for 30 minutes, and rinse with double-distilled water until the eluent becomes transparent. Gels are ready for subsequent analysis.

Bacterial Transformation (e.g., transformation of plasmid containing kanamycin resistance gene into E. coli DH5α)

  1. Thaw a tube of 100μL DH5α competent cells on ice and add 10μL of the plasmid solution. Gently mix cells and DNA by pipetting.
  2. Place the mixture on ice for 30 minutes.
  3. Perform a 42°C heat shock for 90 seconds.
  4. Place the mixture on ice for 2 minutes.
  5. Add 800μL LB culture medium to the mixture and incubate at 37°C in a shaking incubator for 1 hour.
  6. Centrifuge the culture at 12,000 rpm for 5 minutes to concentrate the cells. Remove a certain amount of supernatant and leave 100μL of solution. Gently mix the cells.
  7. Take 100mL of LB solid medium, autoclave, and heat in a microwave oven until the medium is transparent and free of colloidal precipitation.
  8. Place it in a sterile petri dish and add 100μL of kanamycin at a concentration of 50 ng/mL. Shake well and immediately pour into the petri dish. Allow it to solidify.
  9. Divide the 100μL of cells into several portions and add them to the solidified solid medium, spreading them with an applicator. Incubate at 37°C overnight.

Extraction of DNA from Bacterial Cultures

Omega Plasmid Mini Kit I is used to extract DNA from bacterial cultures.

Application object: Used to extract 1-5 mL of E. coli cultured overnight.

Preparing

  1. Heat the Elution Buffer to 70°C if the plasmid DNA is >10kb.
  2. Add a bottle of RNase A to Solution I and store it at 4°C.
  3. Dilute DNA Wash Buffer with 120mL absolute ethanol and store it at room temperature.
  4. Dilute HBC Buffer with 16mL isopropanol.

Steps

  1. Isolate a single colony from a freshly streaked selective plate and inoculate a culture of 1-5 mL LB medium containing the appropriate selective antibiotic. Incubate for 12-16 hours at 37°C with vigorous shaking (220 rpm).
  2. Take the bacterial solution into a new 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 1 minute at room temperature.
  3. Decant or aspirate and discard the culture media. Repeat the operation until all the bacteria are centrifuged.
  4. Add 250μL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of the cell pellet is vital for obtaining good yields.
  5. Add 250μL Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary.
  6. Add 350μL Solution III. Immediately invert several times until a flocculent white precipitate forms. Make sure that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation.
  7. Perform high-speed centrifugation at 14,500 rpm for 10 minutes.
  8. Transfer the supernatant to a HiBind Miniprep DNA binding column with a 2mL collection tube, centrifuge at room temperature for 1 minute, and then pour the filtrate into the collection tube.
  9. Reinstall the adsorption column into the collection tube, add 500μL HBC buffer, centrifuge at room temperature for 1 minute at maximum speed, and then discard the filtrate.
  10. Repeat the above step.
  11. Discard the filtrate and repeat the above step once more.
  12. Discard the filtrate. Transfer the HiBind DNA Mini Column to a 2 mL collection tube. Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix. After centrifugation, transfer the HiBind DNA Mini Column to a clean 1.5 mL microcentrifuge tube, open the lid, and dry at room temperature for three minutes.
  13. Place the adsorption column in a clean 1.5 mL centrifuge tube, add 40μL of elution buffer to the column matrix, let it stand for 1 minute, and centrifuge at 12,000 rpm for 1 minute to elute DNA.
  14. The eluted DNA can be stored at -20°C.

Polymerase Chain Reaction (PCR)

Mix various components before use.

Components (Volume for 50μL Reaction)

  • Q5 High-fidelity 2X Master Mix: 25μL
  • 10μM Forward Primer: 2.5μL
  • 10μM Reverse Primer: 2.5μL
  • Template DNA: 5μL
  • Nuclease-Free Water: To 15μL

Mix the reactants gently and rotate quickly to collect the liquid at the bottom of the PCR tube.

The reaction is quickly transferred to a preheated PCR instrument and operated under the provided thermal cycle conditions.

Touchdown PCR

Mix various components before use.

Components (Volume for 50μL Reaction)

  • Q5 High-fidelity 2X Master Mix: 25μL
  • 10μM Forward Primer: 2.5μL
  • 10μM Reverse Primer: 2.5μL
  • Template DNA: 5μL
  • Nuclease-Free Water: 15μL

Mix the reactants gently and rotate quickly to collect the liquid at the bottom of the PCR tube.

The reaction is quickly transferred to a preheated PCR instrument and operated under the provided thermal cycle conditions.

Restriction Endonuclease Digestion (Double Digestion)

Components (Volume for 50μL Reaction)

  • 10× rCutSmart Buffer: 5μL
  • Not I-HF Restriction Enzyme: 1μL
  • Hind III-HF Restriction Enzyme: 1μL
  • DNA Solution: 1μg (Converted into volume)
  • Deionized Distilled Water: To 50μL
  1. Mix the ingredients on ice.
  2. Incubate the mixture in a thermostatic water bath at 37°C for 15-60 minutes.
  3. Run gel electrophoresis to check the length of the cut DNA fragment.

DNA Ligation

  1. Set the following reaction system in a micro-centrifugal tube on ice.

    2. Components (Volume for 20μL Reaction)

    • T4 DNA Ligase Buffer (10X): 1μL
    • Vector DNA: 50ng (0.020 pmol)
    • Insert DNA: 37.5ng (0.060 pmol)
    • Deionized Distilled Water: To 10μL
    • T4 DNA Ligase: 1μL
  2. The solution is gently mixed and incubated at 16°C for 16 hours or overnight at 4°C.

Induction by IPTG

  1. Select a single colony from the plate and grow it in LB liquid medium containing kanamycin (or streptomycin) at 37°C overnight.
  2. Dilute the overnight culture into 100mL of LB liquid medium and incubate the cells at 37°C until the OD600 reaches 0.6-0.8.
  3. Add 100μL of IPTG (1 mol/L) to the culture and incubate in a shaker at 37°C for 12-16 hours.
  4. Store at 4°C.

Preparation of Competent Cells (Taking E. coli Nissle 1917 as an Example)

  1. Select a single colony from the EcN plate and inoculate it into a shaker tube containing 5mL LB liquid medium. Culture overnight at 37°C.
  2. Transfer 1mL of bacterial culture to an Erlenmeyer flask containing 100mL LB liquid medium and incubate at 37°C for 2-3 hours until the OD600 reaches about 0.4.
  3. Transfer the bacterial culture to a 50mL centrifuge tube that has been sterilized and ice bath for 10 minutes.
  4. Centrifuge the cells at 4°C, 4000 rpm for 10 minutes.
  5. Pour out the medium as much as possible with a pipette.
  6. Suspend the pellet with 10mL of cold 0.1mol/mL CaCl2. Centrifuge at 4°C, 4000 rpm for 1 minute, and then recover the cells.
  7. Suspend the bacterial cells with 2mL of cold 0.1mol/mL CaCl2, gently agitate on ice.
  8. Use the suspension immediately for plasmid transformation or add an equal volume of pre-cooled 60% glycerol solution and store at -80°C for long-term use.

Ultrasonic Crushing of Bacteria

  1. After induction, centrifuge the bacterial culture at 4°C, 4000 rpm for 5 minutes and transfer it to a centrifuge tube.
  2. Discard the supernatant and retain the bacterial pellet. Add 5mL of PBS buffer to resuspend the bacteria.
  3. Centrifuge at 4°C, 4000 rpm for 5 minutes, then discard the supernatant and retain the bacteria.
  4. Repeat the washing step once more.
  5. Add PBS buffer at a ratio of 10mL of buffer to 1g of bacteria to resuspend the bacteria.
  6. Insert the ultrasonic probe and place the surrounding area in ice water. Use the ultrasonic pulverizer for 15 minutes with cycles of 1 second at 100-150W and 2-second rests at 25.2°C.
  7. Centrifuge at 4°C, 12,000 rpm for 20 minutes.
  8. Discard the supernatant. The supernatant can be used for SDS-PAGE or activity detection.