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Plasmid Extraction Protocol

Main materials from Plasmid DNA extraction Micro kit (from ONREW):

Material Amount
Overnight Culture Medium 1-5µL
Buffer S1 250µL
Buffer S2 250µL
Buffer S3 350µL
Buffer W1P 600µL
Buffer W2P 1200µL (two washes of 600µL each)
Buffer EB 50-100µL
  1. Centrifuge Culture:
    • Transfer 1–5 µL of the overnight culture medium to a centrifuge tube and centrifuge at 13,000×g for 1 minute to collect the bacterial pellet.
  2. Remove Culture Medium:
    • Pour off the culture medium and absorb any residual liquid.
    • Add 250 µL of Buffer S1 and vortex at high speed until no bacterial clumps are visible.
  3. Lysis:
    • Add 250 µL of Buffer S2 to the resuspension solution and mix thoroughly by inverting 8–10 times.
  4. Neutralization:
    • Add 350 µL of Buffer S3 and immediately invert the tube 10–15 times to ensure thorough neutralization and full dispersion of the white precipitate.
  5. Centrifuge:
    • Centrifuge at 13,000×g for 10 minutes.
  6. Load Supernatant:
    • Place the DNA Extraction Mini Column I in a collection tube.
    • Transfer the supernatant into the column using a pipette.
    • Centrifuge at 13,000×g for 60 seconds.
  7. Wash with Buffer W1P:
    • Discard the filtrate and place the column back into the collection tube.
    • Add 600 µL of Buffer W1P to the column.
    • Centrifuge at 13,000×g for 60 seconds.
  8. Wash with Buffer W2P:
    • Discard the filtrate and place the column back into the collection tube.
    • Add 600 µL of Buffer W2P (ensure anhydrous ethanol has been added) to the DNA column.
    • Centrifuge at 13,000×g for 60 seconds.
  9. Repeat Wash with Buffer W2P:
    • Discard the filtrate and place the column back into the collection tube.
    • Add 600 µL of Buffer W2P (ensure anhydrous ethanol has been added) to the DNA column.
    • Centrifuge at 13,000×g for 60 seconds.
  10. Dry Column:
    • Discard the filtrate and place the column back into the collection tube.
    • Centrifuge at 13,000×g for 2 minutes to dry the column.
  11. Elute DNA:
    • Place the column in a clean 1.5 µL centrifuge tube.
    • Add 50–100 µL of Buffer EB to the center of the membrane.
    • Let it stand for 2 minutes.
    • Centrifuge at 13,000×g for 1 minute to elute the DNA.
  12. Re-elute DNA:
    • Add the eluate back to the center of the column.
    • Let it stand for 1 minute.
    • Centrifuge at 13,000×g for 1 minute to re-elute the DNA.
  13. Store Plasmid:
    • Discard the column and store the plasmid at -20˚C.

Preparation of Agarose Gel Electrophoresis

Material Amount
Agarose 0.25g
TAE Buffer 25µL
Gel-RED Stain 2.5µL
  1. Prepare Agarose Gel:
    • Weigh 0.25 g of agarose and add 25 µL of TAE buffer.
  2. Dissolve Agarose:
    • Heat the mixture in a microwave oven at medium-high heat for 1 minute.
  3. Cool Mixture:
    • Carefully remove the heated mixture using thick gloves and let it cool down.
  4. Stain Gel:
    • Add 2.5 µL of Gel-RED and mix until a faint pink color is seen.
  5. Pour Gel:
    • Pour the mixture into the electrophoresis gel mold and let it cool and solidify.
  6. Load PCR Product:
    • Add 5 µL of PCR product or DNA marker to the wells of the agarose gel.
  7. Run Electrophoresis:
    • Cover the gel with the electrophoresis lid and apply 120V for 30 minutes.
  8. Visualize Gel:
    • Place the finished agarose gel in a gel imager and turn on the UV lamp.
  9. Analyze Results:
    • Observe and compare the DNA sizes using a marker and save the image.

PCR Protocol

PCR system (50µL system):

Material Amount
Template 1µL
Primer - F 1µL
Primer - R 1µL
2×Super Pfx Master Mix 25µL
ddH2O 22µL (to fill up the system)
  1. PCR Set up (50µL system):
    • In a PCR tube, add 1µL of your template.
    • Each of the Primer -F and Primer – R, add 1µL.
    • Add 25µL of your Mix to the mixture.
    • Fill up the system to 50µL by adding ddH2O.
  2. PCR Setting:
    • Put your PCR tube into a thermal cycler.
    • Heat at 98°C for 5 minutes to denature DNA.
    • Continue denaturation by heating for 30 seconds at 98°C.
    • Decrease the temperature to 50-60°C (The exact temperature is determined by the primer Tm value) and incubate for 30 seconds to allow primers to bond to the single-stranded DNA.
    • Heat to 72°C for 1 minute (The exact duration is determined by the length of the fragment to be amplified and the capacity of the DNA polymerase). Taq polymerase joins deoxyribonucleotides to extend the DNA strand.
    • Repeat steps 2 to 4 for a total of 30 cycles.
    • Perform a final extension at 72°C for 5 minutes to ensure complete extension of all DNA fragments.
    • Store the PCR product at 4°C.

Restriction Enzyme Digestion

For Plasmid DNA (10 µL System):

Material Amount
Plasmid DNA (50-100 ng/µL) 5µL
First restriction enzyme 1µL
Second restriction enzyme 1µL
10×Buffer 1µL
ddH2O To fill up the system
  1. Prepare Reaction Mixture:
    • Add 5 µL of plasmid DNA to a PCR tube.
    • Add 1 µL of the first restriction enzyme to it.
    • Add 1 µL of the second restriction enzyme.
    • Add 1 µL of buffer.
    • Add ddH2O to bring the total volume to 10 µL.
  2. Mix and Incubate:
    • Gently mix the components in a PCR tube.
    • Place the PCR tube in a thermal cycler at 37°C for 1 hour.

For Target DNA (10 µL System):

Material Amount
Target DNA (50-100 ng/µL) 5µL
First Restriction Enzyme 1µL
Second Restriction Enzyme 1µL
Buffer 1µL
ddH2O To fill up the system
  1. Prepare Reaction Mixture:
    • Add 5 µL of target DNA.
    • Add 1 µL of the first restriction enzyme.
    • Add 1 µL of the second restriction enzyme.
    • Add 1 µL of buffer.
    • Add ddH2O to bring the total volume to 10 µL.
  2. Mix and Incubate:
    • Gently mix the components in a PCR tube.
    • Place the PCR tube in a thermal cycler at 37°C for 1 hour.
Image 1
Image 2

T4 DNA Ligase Reaction (10 µL System)

Material Amount
Plasmid DNA 3µL
Target DNA 5µL
T4 DNA Ligase 1µL
Ligase Buffer 1µL
  1. Prepare Ligation Mixture:
    • Add 5 µL of the target DNA product.
    • Add 3 µL of the carrier DNA product.
    • Add 1 µL of Ligase buffer.
    • Add 1 µL of T4 DNA ligase.
  2. Mix and Incubate:
    • Gently mix the components in a PCR tube.
    • Place the tube in a thermal cycler set at 22°C for at least 10 minutes.

Gibson Assembly

Construct plasmid using the target gene and the vector using Gibson assembly.

Material Amount
Sterilized water Sufficient enough to add to 10µL
2x CE Mix 5µL
Vector sample Calculate according to instructions
Target gene sample Calculate according to instructions
  1. Calculate the sample volumes of the target gene and the vector according to the formulas:
    • Target gene volume = bp x 0.04 / concentration
    • Vector volume = bp x 0.02 / concentration
  2. Calculate the volume of sterilized water required to make up to 10 μl.
  3. Add the sterilized water, 2x CE Mix, vector sample and Target gene sample into the EP tube.
  4. Put the EP tube in a centrifuge for centrifugation.
  5. Put the EP tube in the PCR instrument, set it to 50˚C for 15 minutes.

DNA Purification Protocol

TIAN quick MIDI purification kit (from TIANGEN):

Material Amount
Equilibration Buffer (BL) 500µL per sample
Binding Buffer (PB) 5x the volume of PCR reaction mixture
Wash Buffer (PW) 1200µL, 600/wash
Elation Buffer (EB) 30-µL per sample
  1. Equilibrate Column:
    • Add 500 µL of Equilibration Buffer (BL) to the adsorption column CB2 (place the adsorption column into a collection tube).
    • Centrifuge at 12,000 rpm (~13,400×g) for 1 minute.
    • Discard the waste liquid from the collection tube and place the adsorption column CB2 back into the collection tube.
  2. Bind DNA:
    • Add 5 volumes of Binding Buffer (PB) to the PCR reaction mixture or enzyme digestion mixture and mix well (no need to remove paraffin oil or mineral oil).
  3. Load Solution:
    • Add the mixture from the previous step to an adsorption column CB2 (place the adsorption column into a collection tube).
    • Let it sit at room temperature for 2 minutes.
    • Centrifuge at 12,000 rpm (~13,400×g) for 30-60 seconds.
    • Discard the waste liquid from the collection tube and place the adsorption column CB2 back into the collection tube.
  4. First Wash:
    • Add 600 µL of Wash Buffer (PW) (ensure anhydrous ethanol has been added before use) to the adsorption column CB2.
    • Centrifuge at 12,000 rpm (~13,400×g) for 30-60 seconds.
    • Discard the waste liquid from the collection tube and place the adsorption column CB2 back into the collection tube.
  5. Second Wash:
    • Repeat step 4.
  6. Dry:
    • Place the adsorption column CB2 back into the collection tube.
    • Centrifuge at 12,000 rpm (~13,400×g) for 2 minutes to remove the wash buffer as much as possible.
    • Let the adsorption column CB2 air dry at room temperature for several minutes to prevent residual wash buffer from affecting the next experiment.
  7. Elute:
    • Place the adsorption column CB2 into a clean centrifuge tube.
    • Add 30-50 µL of Elution Buffer (EB) to the center of the adsorption membrane.
    • Let it sit at room temperature for 2 minutes.
    • Centrifuge at 12,000 rpm (~13,400×g) for 2 minutes to collect the DNA solution.

Agarose Gel DNA Recovery

TIAN gel MIDI purification Kit (from TIANGEN):

Material Amount
Equilibration Buffer (BL) 500µL
PN Solution Equal to gel slice volume
Wash Buffer (PW) 1200µL, 600/wash
Elation Buffer (EB) 30-50µL per sample
  1. Column Equilibration Step:
    • Add 500 µL of Equilibration Buffer (BL) to the adsorption column CA2 (place the adsorption column into a collection tube).
    • Centrifuge at 12,000 rpm (~13,400×g) for 1 minute.
    • Discard the waste liquid from the collection tube and place the adsorption column CA2 back into the collection tube.
  2. Excise DNA Band:
    • Cut the single desired DNA band from the agarose gel (remove as much excess gel as possible) and place it into a clean centrifuge tube.
    • Weigh the gel slice.
  3. Dissolve Gel Slice:
    • Add an equal volume of PN solution to the gel slice (e.g., if the gel weighs 0.1 g, its volume can be considered as 100 µL, then add 100 µL PN solution).
    • Incubate at 50°C in a water bath, gently inverting the centrifuge tube to ensure the gel slice is fully dissolved.
    • If any gel pieces remain undissolved, continue incubating for a few more minutes or add more PN solution until the gel is completely dissolved.
  4. Transfer Solution to Adsorption Column:
    • Add the solution from the previous step to an adsorption column CA2 (place the adsorption column into a collection tube).
    • Let it sit at room temperature for 2 minutes.
    • Centrifuge at 12,000 rpm (~13,400×g) for 30-60 seconds.
    • Discard the waste liquid from the collection tube and place the adsorption column CA2 back into the collection tube.
  5. Wash Column:
    • Add 600 µL of Wash Buffer (PW) to the adsorption column CA2.
    • Centrifuge at 12,000 rpm (~13,400×g) for 30-60 seconds.
    • Discard the waste liquid from the collection tube and place the adsorption column CA2 back into the collection tube.
  6. Repeat Wash:
    • Repeat step 5.
  7. Dry Column:
    • Place the adsorption column CA2 back into the collection tube.
    • Centrifuge at 12,000 rpm (~13,400×g) for 2 minutes to remove the wash buffer as much as possible.
    • Let the adsorption column CA2 air dry at room temperature for several minutes to prevent residual wash buffer from affecting the next experiment.
  8. Elute DNA:
    • Place the adsorption column CA2 into a clean centrifuge tube.
    • Add an appropriate amount of Elution Buffer (EB) to the center of the adsorption membrane.
    • Let it sit at room temperature for 2 minutes.
    • Centrifuge at 12,000 rpm (~13,400×g) for 2 minutes to collect the DNA solution.

Colony PCR

PCR system (A 10µL system):

Material Amount
Single Colonies Appropriate
Primer - F 0.1µL
Primer - R 0.1µL
2×Rapid Taq Master Mix 5µL
ddH2O To fill up the system
  1. Preparation of PCR Products:
    • Dispense 10 µL of PCR systems into each of 10 PCR tubes using a pipette.
  2. Control Tube Preparation:
    • Positive Control (Tube A) - Add 0.2 µL template DNA (PCR product) to Tube A.
    • Negative Control (Tube B) - Add E. coli without target gene to Tube B.
  3. Single Colony PCR Preparation:
    • Single Colony Addition: For the remaining 8 tubes, add a single colony to each tube.

Bacteria Inoculation

60µL System:

Material Amount
LB Medium 5µL
E. Coli Solution 50µL
Antibiotics 5µL
  1. Disinfect Biosafety Cabinet:
    • Turn on the UV light to disinfect the biosafety cabinet.
  2. Prepare Biosafety Cabinet:
    • Turn off the UV light, lift the glass shield, and turn on the illumination and ventilation.
  3. Disinfect Hands and Equipment:
    • Use 75% ethanol to disinfect hands and laboratory equipment.
  4. Prepare Culture Medium:
    • Add 5 µL of LB medium to a 50 µL centrifuge tube.
  5. Add Bacterial Solution:
    • Add 50 µL of E. coli solution to the centrifuge tube.
  6. Add Antibiotics:
    • Add 5 µL of antibiotics to the centrifuge tube.
  7. Incubate:
    • Place the centrifuge tube in a shaker at 37°C, 200 RPM, and incubate overnight.

Bacteria Preservation

Material Amount
50% concentration glycerol : Bacterial solution 1:1
  1. Disinfect Biosafety Cabinet:
    • Turn on the UV light to disinfect the biosafety cabinet.
  2. Prepare Biosafety Cabinet:
    • Turn off the UV light, lift the glass shield, and turn on the illumination and ventilation.
  3. Prepare Glycerol Stock:
    • Mix glycerol of concentration at 50% and the bacterial solution in a 1:1 ratio and place the mixture in an EP tube.
  4. Storage:
    • Store the EP tube at -20°C.

Spectrophotometric Detection

  1. Turn on the spectrophotometer power and wait for the initialization to end.
  2. Click to move the wavelength and enter the detection wavelength.
  3. Add the blank control to the cuvette, put it in the slot, close the light shield cover, move the pull rod to the blank control, and press “100% OA” to zero.
  4. Put the sample into the cuvette, close the light shield cover, and move the pull rod to the sample.
  5. After the detection is completed, take out the cuvette and clean it with distilled water.
  6. Turn off the power to the spectrophotometer.

Preparation and Transformation of Competent Yeast Cells

Material Amount
Liquid Culture Medium 5ml
ddH2O 27ml
Salmon Sperm DNA 10µL
PEG3350 (50% (w/v)) 240µL
LiAc (1.0M) 36µL
Plasmid DNA 2-5µL/transformation
Sterile Water To fill up the system
YPD Liquid Medium 1mL
  1. Inoculate Yeast:
    • Use a sterile inoculating loop to transfer a single yeast colony from a fresh YPAD plate to 5 mL of liquid culture medium (YPAD or SC selective medium).
    • Incubate with shaking at 200 rpm, 30°C overnight.
    • Preheat a bottle of 250 mL 2 YPAD medium in a 30°C incubator.
    • After 12-16 hours, measure the concentration of the yeast culture.
  2. Measure OD600:
    • Pipette 10 µL of the yeast cells into 1mL of water in a cuvette for a spectrophotometer, mix thoroughly.
    • Measure the OD value at 600 nm (OD600).
    • Multiply by the dilution factor to determine the titer of the cell culture (an OD600 of 0.1 corresponds to a suspension of 1 106 cells/mL).
  3. Inoculate Preheated Medium:
    • Transfer 2 mL of the bacterial solution into preheated 2*YPAD medium.
    • Incubate with shaking at 200 rpm, 30°C for about 4-6 hours until the cell concentration reaches OD600 approximately equal to 0.8.
  4. Harvest Cells:
    • Centrifuge at 3,000g for 5 minutes to harvest the cells.
    • Resuspend the cell pellet in 25 mL of sterile water, repeat this step 3 times.
    • Resuspend the final pellet in 1 mL of sterile water.
  5. Prepare Carrier DNA:
    • Denature 10 µL of salmon sperm DNA by boiling for 5 minutes, then cool immediately in an ice/water bath.
  6. Prepare Aliquots:
    • Transfer the cell suspension to 1.5 mL micro-centrifuge tubes, aliquot 100 µL per tube.
    • Centrifuge at 13,000g for 30 seconds, discard the supernatant.
  7. System:
    • PEG 3350 (50% (w/v)): 240 µL, LiAc (1.0 M): 36 µL, Plasmid Vector DNA: 2-5 µL, Salmon Sperm DNA: 10 µL
    • Sterile water: add to bring the total volume to 360µL.
  8. Add Transformation Mixture:
    • Add 360 µL of the transformation mixture to each tube.
    • Vortex to resuspend the cells thoroughly, including a negative control tube without plasmid DNA.
    • Incubate the tubes in a 42°C water bath for 40 minutes.
  9. Post-transformation Incubation:
    • Centrifuge at 13,000g for 30 seconds, remove the supernatant.
    • Add 1.0 mL YPD liquid medium to the transformation tube.
    • Vortex to resuspend the pellet, then incubate at 30°C for 2-3 hours.
  10. Plate Cells:
    • Spread 2 µL, 20 µL, or 200 µL of the cell suspension on appropriate SC selective medium plates.

Yeast Protein Extraction Protocol

Main materials from Protein Extraction:

Material Amount
Yeast Culture Liquid 5ml
Isotonic Fluids 3.7ml
Snail Enzymes 20μl
β-mercaptoethanol 4μl
Hypotonic Fluid 2ml
Omni-Easy™ Protein Sample Loading Buffer (4×) 5μl
  1. Preparation for the Protein Extraction:
    • Pour the 72-hour culture into an EP tube.
    • Centrifuge at 8,000 rpm (~5,000g) for 1 min, discard the waste liquid from the EP tubes.
  2. Weigh the Saccharomyces Cerevisiae
  3. Add Isotonic Fluids, Snail Enzymes and β-mercaptoethanol into the EP Tubes:
    • Add 10μl of isotonic solution per mg of bacterium.
    • Add 5μl snail enzymes into all the tubes.
    • Add 1μl β-mercaptoethanol into all the tubes.
  4. Break the Cell Wall:
    • Repeatedly blow the bacterial solution with a pipette gun to resuspend the bacterial cells and vortex the tubes.
    • Place the EP tubes into a thermal cycler set at 30℃ for 60 min.
    • Centrifuge at 5,000 rpm (~2,500g) for 1 min, discard the waste liquid from the EP tubes.
    • Add 500μl isotonic fluids into all the EP tubes, resuspend the bacterial cells and vortex the tubes.
    • Centrifuge at 5,000 rpm (~2,500g) for 1 min, discard the waste liquid from the EP tubes.
    • Repeat the last two steps.
    • Add 500μl hypotonic fluid into all EP tubes, resuspend the bacteria, and vortex the tubes.
    • Put all the EP tubes into -20 ℃ fridge for 45 min, then move them back to room temperature for dissolving.
    • Repeat the last step for two times.
  5. Collect the Protein:
    • Centrifuge at 12,000 rpm (~12,830g) for 5 min, discard the waste liquid from the EP tubes.
  6. Store the Protein:
    • Put the EP tubes into -20 ℃ fridge for storing.

SDS-PAGE (Poly Acry Lamide Gel Electrophoresis) Protocol

Main materials from SDS-PAGE:

Material Amount
Beyo GeI™ SDS-PAGE Precast Gel 10%
Tris-Glycine Fulfil the inner portion and outer chamber
ddH2O 150ml
Coomassie Blue 20ml
Boiling Water 150ml
Protein Marker 10μl
Protein Standard 10μl
  1. Instrument Preparation:
    • Decide how many percentages of gel used to separate proteins:
    • Beyo GeI™ SDS-PAGE Precast Gel, 10%, for 20-80kD.
    • Take off the orange paper of the bottom of the precast gel and place the Beyo GeI™ SDS-PAGE Precast Gel into a clean plastic electrophoresis chamber, correspond the holder.
    • Fill the inner portion and outer chamber with 10*Tris-Glycine.
  2. Protein Preparation:
    • Put six micro centrifuge tubes into a water bath (set to 100℃) for 10 min.
  3. Protein Electrophoresis:
    • Load four samples into gel lanes starting with markers. The number of the samples should all be 20μl, while the marker and the protein standard should be 10μl.
    • Cover the chamber and connect the anode and the cathode. Set the voltage on the electrophoresis power supply to a constant voltage of 80V, wait for 50 min.
    • Then change the voltage to 120V, wait for 40 min.
    • Turn off the power immediately when the dye front migrates out from the bottom of the gel.
    • Disconnect the electrodes and remove the cover. Take the gel holder out from the electrophoresis chamber. Use the comb to carefully remove the gel from the holder and place it into a plate.
  4. Washing:
    • Add 50ml ddH2O, then heat the mixture in a microwave oven at high heat for 3 min.
    • Shake the plate for 5 min, then pour out the water.
    • Repeat washing for another 2 times.
  5. Coomassie Blue Staining:
    • Add 20ml Coomassie Blue, then heat the plate in a microwave oven at medium-high heat for 3 min.
    • Place the plate into a shaker for 30 min, then pour out the water.
  6. Decolorizing:
    • Add 50ml boiling water into the plate.
    • Shake the plate for 5 min, then pour out the water.
    • Repeat the decolorizing for another 2 times.

N-Acetylneuraminic Acid Assay

The experiment involves four tubes: a blank tube for blank control, a standard tube for positive control, a culture medium tube for negative control, and the measuring tubes for measuring the content of N-Acetylneuraminic acid.

Material Blank Tube Standard Tube Measuring Tube
Distilled Water (mL) 0.1
1mol/L sialic acid solution (mL) 0.1
Solutions that need to be measured (mL) 0.1
S1 (mL) 0.2 0.2 0.2
S2 - Chromogenic agent (mL) 4.0 4.0 4.0
  1. Mix the components.
    Image 1
    Image 2
  2. 100℃ water bath for 15 minutes.
  3. After cooling down, centrifuge at 3,000~3,500 RPM at room temperature for 10 minutes.
  4. Take the supernatant of each tube.
  5. Use a spectrophotometer to measure the absorbance value of each tube, at 560nm wavelength, 1cm light diameter.
  6. Zero setting by 2mL water before the operation.

The absorbance value collected from the spectrophotometer can be used to calculate the content of sialic acid in the sample with the following equation:

Content of sialic acid (mmol/L) = \[ \frac{A_{\text{measure}} - A_{\text{blank}}}{A_{\text{standard}} - A_{\text{blank}}} \times C_{\text{standard}} \]

Cstandard = the concentration of the standard tube.

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