Bacterial Transformation Protocol (E. coli) ​

Materials:

• Competent E. coli cells
• Liquid/solid LB medium
• Appropriate antibiotic (confirm plasmid resistance in advance - Kanamycin)

Procedure:

1. Thaw competent cells on ice for 5 to 10 minutes.
2. Add 100 µL of competent cells to a pre-chilled 1.5 mL microcentrifuge tube.
3. Add approximately 5 ng (typically 5-10 μL) of plasmid DNA to the competent cells. Gently mix by flicking or inverting the tube 4-5 times. Do not vortex.
4. Place the mixture on ice for 30 minutes.
5. Heat shock at 42°C for exactly 30 seconds. Do not mix.
6. Place on ice for 2-3 minutes.
7. Add 900 µL of room temperature LB medium to the tube.
8. Incubate the tube at 37°C for 60 minutes with vigorous shaking (220 rpm).
9. Pre-warm selective plates to 37°C.
10. Spread 25-50 µL of the cell and ligation mixture onto the Petri dish. (Add some Kanamycin).
11. Incubate overnight at 37°C.

Plasmid Extraction and Vector Selection (miniprep) ​

Objective: To extract plasmid DNA (containing the gene of interest) from positive bacterial colonies for use as a template in subsequent PCR.

Materials:

• Competent cells
• Liquid/solid LB medium
• Corresponding antibiotic (confirm plasmid resistance - Ampicillin/Kanamycin)
• Miniprep kit

Protocol:

Preparation:

• Check Reagents: Ensure RNase A is added to Buffer SP1, and absolute ethanol is added to Wash Solution. Check that there is no precipitation in Buffer SP2 and SP3.

Column Equilibration:

• Add 500 µL of Buffer S to the adsorption column.
• Centrifuge at 12,000 × g for 1 minute.
• Discard the flow-through and place the adsorption column back into the collection tube.

Bacterial Pellet Collection:

• Transfer 1.5 - 5 mL of the overnight bacterial culture to a microcentrifuge tube.
• Centrifuge at 8,000 × g for 2 minutes.
• Completely discard the supernatant (culture medium).

Cell Resuspension:

• Add 250 µL of Buffer SP1 to the pellet and resuspend thoroughly.

Lysis and Neutralization:

• Add 250 µL of Buffer SP2 and gently invert 5-10 times to mix.
• Let the mixture stand at room temperature for 2-4 minutes.
• Add 350 µL of Buffer SP3 and gently invert 5-10 times to mix.
• Centrifuge at 12,000 × g for 5-10 minutes.
• Transfer the supernatant to the adsorption column and centrifuge at 8,000 × g for 30 seconds.

Optional Step:

• Add 500 µL of Buffer DW1 and centrifuge at 9,000 × g for 30 seconds.

Washing:

• Add 500 µL of Wash Solution and centrifuge at 9,000 × g for 30 seconds.
• Repeat this step once.

Elution:

• Empty spin: Centrifuge the adsorption column at 9,000 × g for 1 minute.
• Place the adsorption column into a new, sterile microcentrifuge tube.
• Add 50-100 μL of Elution Buffer to the center of the column.
• Let stand at room temperature for 1 minute.
• Centrifuge for 1 minute.
• Save the resulting DNA solution in the tube.

Quantitative Plasmid DNA Purification (Nanodrop) ​

Introduction: Absorbance measurements, as performed on a spectrophotometer like the Thermo NanoDrop One, will include all molecules in a sample that absorb light at the wavelength of interest. This type of spectrophotometer is designed to measure the concentration and purity of nucleic acids and proteins using a very small volume. The ratio of absorbance at 260 nm to 280 nm is used to evaluate the purity of DNA and RNA. A ratio of 1.8 is generally accepted as the "pure" standard for DNA.

Equipment:

• NanoDrop One Spectrophotometer
• Micropipette and tips

Procedure:

1. Turn on the NanoDrop One. Allow the instrument to warm up and initialize.
2. Raise the arm and clean both pedestals with a new laboratory wipe (Kimwipes). Add 3 µL of deionized water to the lower pedestal. Lower the arm and wait for a moment. Raise the arm and clean both pedestals with a laboratory wipe.
3. From the main screen, select the Nucleic Acids tab and click on dsDNA.
4. Blank: Add 2 µL of Elution Buffer (EB) from the first part of the protocol to the lower pedestal and lower the arm. Tap Blank and wait for the measurement to complete. (This step ensures the spectrophotometer is calibrated to the solvent, and that its absorbance is not included in the final DNA measurement).
5. Raise the arm and clean both pedestals with a laboratory wipe.
6. Pipette 2 µL of your first plasmid DNA sample onto the lower pedestal and lower the arm. Once the sample measurement is complete, the spectrum and reported values will be displayed.
7. Raise the arm and clean both pedestals with a laboratory wipe.
8. Using a pipette, transfer 2 µL of your second plasmid DNA sample onto the lower pedestal and lower the arm. Once the sample measurement is complete, the spectrum and reported values will be displayed.
9. Fill out the provided table, noting the reported values. Calculate the required volume for 1 µg (=1000 ng) and 500 ng of DNA.
10. After measuring all your samples, tap End Experiment.
11. Pipette 3 µL of deionized water onto the lower pedestal and lower the arm. Raise the arm and clean both pedestals with a laboratory wipe.

P.S.: The NanoDrop One will calculate the DNA concentration based on the known absorption coefficient for double-stranded DNA (50 µg/mL for every absorbance unit at 260 nm). Additionally, the NanoDrop software provides the ratio of absorbance at 260 nm to that at 280 nm (A260/A280) and the ratio of absorbance at 260 nm to that at 230 nm (A260/A230) to evaluate the purity of the DNA. A typical A260/A280 ratio for pure DNA is 1.8, and the A260/A230 ratio is 2.0-2.2.

PCR Amplification of Gene of Interest (Primers) ​

Objective: To amplify the gene of interest from the extracted plasmid, using it as a template.

Materials:

• PCR Kit (containing high-fidelity enzyme, dNTPs, primers)
• PCR Thermocycler

Key Points:

• Confirm PCR reaction conditions in advance (e.g., annealing temperature, number of cycles).
• Primers must be a match for the gene of interest sequence.

Relevant DNA Fragment Sizes and Temperatures:

Colony PCR Protocol ​

Materials:

• 2x FidCycle Evo PCR Master Mix
• Colonies from liquid cultures
• Forward primer and reverse primer (10 µM)

Procedures:

1. Pick a colony and place it into a 200 µL PCR tube containing 10 µL of water (colony tube), and incubate at room temperature for 30 minutes.
2. Assemble all reaction components in PCR tubes. The components used for PCR are shown in Table 3.
3. Perform PCR using the touchdown PCR protocol.

Table 3. Components of cPCR reaction system.

Touchdown PCR ​

1. Assemble all reaction components in a PCR tube. Assembly should be done on ice. The components for the PCR are shown in Table 1.
2. Collect all liquid to the bottom of the tube by centrifuging for a few seconds.
3. Place the PCR tube into the thermocycler and run the following program (Touchdown PCR).

Note: The total number of cycles is between 35-40. It can be less, but should not exceed this range (Senior student Xu usually uses 10+30 cycles). We use 2x FidCycle Evo PCR Master Mix.

Table. Components of PCR mixture

PCR Program:

• Initial Denaturation Step:
  • Temperature: 98°C
  • Time: 10 minutes
  • Purpose: To completely denature the template DNA.
• Touchdown Cycles (e.g., for 10 rounds):
  • Denaturation: 95°C for 30 seconds
  • Annealing: Start at 65°C, decrease by 1°C per round
  • Extension: 72°C, 30s/kb (adjust based on amplified fragment size)
• Standard PCR Cycles (e.g., for another 25 rounds):
  • Denaturation: 95°C for 30 seconds
  • Annealing: 55°C for 30 seconds
  • Extension: 72°C, 30s/kb
• Final Extension Step:
  • Temperature: 72°C
  • Time: 10 minutes
  • Purpose: To ensure all fragments are fully extended.
• Hold or Storage:
  • Temperature: 4°C or room temperature.

Melatonin (MT) ELISA Kit ​

Operating Procedure

Restore all reagents and components to room temperature before use. It is recommended to run standards, quality control samples, and test samples in duplicate.

1. Prepare the working solutions of all kit components as described in the previous instructions.
2. Take out the required microplate strips from the aluminum foil pouch; seal and return the unused strips to the refrigerator. Set up the standard wells, blank wells, and sample wells.
   • Add 50µL of different concentrations of standard to the standard wells.
   • The blank wells remain empty.
   • Add 50µL of test sample to the sample wells.
3. Add 100µL of horseradish peroxidase (HRP) labeled detection antigen to the standard wells and sample wells (excluding the blank wells).
4. Cover the reaction plate with a sealing film and incubate in a 37°C water bath or constant temperature incubator for 60 minutes.
5. Remove the sealing film, discard the liquid, and pat dry on absorbent paper.
   • Fill each well with wash buffer, let stand for 20 seconds, shake out the wash buffer, and pat dry on absorbent paper.
   • Repeat this step 5 times.
   • If using an automatic plate washer, follow the washer's operating procedure and add a 30-second soaking step to improve detection accuracy.
   • After washing and before adding the substrate, the reaction plate must be thoroughly patted dry on a clean, lint-free paper.
6. Thoroughly mix Substrate A and B at a 1:1 volume ratio. Add 100µL of the mixed substrate solution to all wells.
7. Cover the reaction plate with a sealing film and incubate in a 37°C water bath or constant temperature incubator for 15 minutes.
8. Add 50µL of stop solution to all wells. Read the absorbance (OD value) of each well on the microplate reader.

Use the microplate reader at a wavelength of 450nm. Zero the instrument using the blank control well before measuring the optical density (OD value) of all other wells.

Calculation of Results

1. After the detection is complete, use standard concentration as the Y-axis and the corresponding absorbance (OD value) as the X-axis.
2. Use computer software to create a standard curve equation by employing a four-parameter Logistic curve fitting (4-pl).
3. Calculate the sample concentration value by using the sample's absorbance (OD value) with the standard curve equation.
4. If the sample was diluted, the concentration value calculated must be multiplied by the dilution factor to obtain the final concentration of the sample.

Serotonin ELISA kit ​

Materials and Preparation

Reagents and Equipment:

• The kit includes a pre-coated microplate, standards (800, 400, 200, 100, 50, and 0 ng/mL), SA-HRP, Assay Diluent, TMB substrate, stop solution, and a 20x concentrated wash solution.
• Required external equipment includes an ELISA plate reader (450 nm), precision pipettes, a 37°C water bath or incubator, distilled water, and a wash bottle or automated plate washer.

Sample Handling:

• Samples can be serum, plasma, cell culture supernatant, or tissue.
• For serum and plasma, centrifuge the sample at 4000 rpm for 20 minutes to get the supernatant.
• Tissue samples should be rinsed with pre-cooled PBS, weighed, and then homogenized with PBS (usually a 1:9 weight-to-volume ratio). The homogenate should then be centrifuged at 5000×g for 5-10 minutes to obtain the supernatant for testing.
• All samples should be stored at -20°C or below and should avoid repeated freeze-thaw cycles.

Assay Protocol

1. Move all reagents to room temperature for two hours. Prepare the 1x wash solution by diluting the 20x concentrate with distilled water at a 1:20 ratio.
2. Take the pre-coated plate out of the sealed bag.
3. Set up one blank control well (add no liquid).
4. Add 15 µL of each standard to its corresponding duplicate wells.
5. Add 15 µL of the test sample or QC sample to each remaining well.
6. Add 100 µL of Assay Diluent to all wells except the blank control. Mix and seal the plate with a membrane.
7. Incubate the plate at 37°C for 60 minutes.
8. Wash the plate manually or with a plate washer. For manual washing, discard the liquid, fill each well with wash solution, let stand for 10 seconds, shake dry, and repeat 3 times.
9. Add 100 µL of SA-HRP to all wells except the blank. Mix, seal the plate, and incubate at 37°C for 30 minutes.
10. Wash the plate three times.
11. Add 100 µL of TMB to each well. Shake to mix and incubate at 37°C in the dark for 15 minutes.
12. Add 100 µL of Stop Solution to each well.
13. Read the OD value at 450 nm using an ELISA reader. Use the blank control to zero the instrument before reading other wells.

Superoxide Dismutase (SOD) Activity Assay Kit (Micro Method) ​

Kit Components

Required but Not Provided Materials

• Visible Spectrophotometer / ELISA reader
• Benchtop centrifuge
• Adjustable pipette
• Micro glass cuvette / 96-well plate
• Mortar / Homogenizer
• Ice and distilled water

Sample Handling

• Cells/Bacteria: Collect bacteria or cells into a centrifuge tube, discard the supernatant after centrifugation. Add 1 mL of extraction solution for every 5 million bacteria or cells. Sonicate (20% power or 200W, sonicate for 3s, interval 10s, repeat 30 times). Centrifuge at 8000g at 4°C for 10 minutes, take the supernatant, and place it on ice for testing.
• Tissue Samples: Weigh about 0.1 g of tissue, add 1 mL of extraction solution, and homogenize in an ice bath. Centrifuge at 8000g at 4°C for 10 minutes, take the supernatant, and place it on ice for testing.
• Serum/Plasma Samples: Test directly.

Operational Steps

• Before the formal test, it is essential to perform a preliminary test with 2-3 samples with a large expected difference.

1. Preheat the spectrophotometer / ELISA reader for more than 30 minutes, set the wavelength to 560 nm, and zero the instrument with distilled water.
2. Before measurement, preheat Reagents 1, 3, and 4 in a 37°C (for mammals) or 25°C (for other species) water bath for at least 5 minutes.
3. Add reagents sequentially according to the following table:

4. Mix thoroughly, incubate in a 37°C water bath for 30 minutes, then measure the absorbance (A) of each tube at 560 nm. If there is a precipitate at the bottom, mix again before measurement.

Activity Calculation

1. Calculation of Inhibition Percentage:

• Inhibition % = (ΔAblank - ΔAtest) / ΔAblank × 100%
• Where ΔAtest = Atest - Acontrol and ΔAblank = Ablank1 - Ablank2.
• Try to keep the sample's inhibition percentage within the 30-70% range; the closer to 50%, the more accurate the result.

2. SOD Enzyme Activity Unit:

• An SOD activity unit is defined as the amount of SOD that causes a 50% inhibition in the xanthine oxidase-coupled reaction system.

3. SOD Enzyme Activity Calculation:

• Serum/Plasma SOD activity (U/mL):SOD activity = 10 × Inhibition % / (1 - Inhibition %) × F
• Tissue, bacteria, or cultured cells SOD activity:
  • By protein concentration (U/mg prot):SOD activity = 10 × Inhibition % / [(1 - Inhibition %) × Cpr] × F
  • By sample weight (U/g weight):SOD activity = 10 × Inhibition % / [(1 - Inhibition %) × W] × F
  • By cell/bacteria count (U/10^6 cells):SOD activity = 0.022 × Inhibition % / (1 - Inhibition %) × F

Note:

• Vreactiontotal = 0.2 mL
• Vsample = 0.02 mL
• Vextractiontotal = 1 mL
• Cpr = protein concentration (mg/mL)
• W = sample weight (g)
• F = sample dilution factor
• 500 = total number of cells or bacteria (5 million)

Precautions

1. Samples and Reagent 2 should be kept on ice during use.
2. If you have many samples, you can prepare a working solution that includes Reagents 1, 2, and 3. Reagent 4 must be added last.
3. After the reaction is complete, a precipitate may form. Mix before measuring.

Peroxidase (POD) Activity Assay Kit: Colorimetric Method ​

Detection Principle POD (EC 1.11.1.7) is widely present in animals, plants, and microorganisms. It catalyzes the oxidation of phenolic and amine compounds by hydrogen peroxide, thereby eliminating the toxicity of both hydrogen peroxide and the phenolic/amine compounds. In the presence of hydrogen peroxide, POD oxidizes guaiacol to form a tea-brown substance which has a maximum light absorption at 470nm.

Kit Components

Materials Required but Not Provided Visible spectrophotometer / Microplate reader, desktop centrifuge, adjustable pipettes, micro glass cuvettes / 96-well plates, mortar and pestle / homogenizer, ice, and distilled water.

Sample Preparation

1. Bacteria or Cells:
   • Collect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant.
   • Add 1mL of extraction buffer for every 5 million bacteria or cells.
   • Sonicate the bacteria or cells (power 20%, 3s sonication, 10s interval, repeat 30 times).
   • Centrifuge at 8000g at 4°C for 10min.
   • Take the supernatant and keep it on ice for testing.
2. Tissue Samples:
   • Weigh approximately 0.1g of tissue.
   • Add 1mL of extraction buffer and homogenize in an ice bath.
   • Centrifuge at 8000g at 4°C for 10min.
   • Take the supernatant and keep it on ice for testing.
3. Serum (Plasma) Samples: Directly test the sample.

Operating ProcedureNote: Before formal testing, it is essential to perform a pre-test with 2-3 samples expected to have large differences.

1. Preheat the spectrophotometer or microplate reader for more than 30min. Set the wavelength to 470nm and zero the instrument with distilled water.
2. Place Reagent I, II, and III at 37°C (mammals) or 25°C (other species) for more than 10min before measurement.
3. Add the reagents sequentially according to the table below:

4. Add the reagents in sequence to an EP tube. Immediately mix and start timing.
5. Immediately transfer 200µL to a micro glass cuvette or 96-well plate.
6. Record the absorbance at 470nm at 30s (Absorbance A1) and after 1min30s (Absorbance A2).
7. Calculate the change in absorbance: ΔA=A2-A1.

Note: If a large number of samples are being tested at once, Reagents I, II, III, and distilled water can be mixed proportionally, incubated for 10min at 37°C (mammals) or 25°C (other species), and then 240µL of the mixture can be added for the assay.

Activity Calculation

a. Using Micro Glass CuvettesUnit Definition: One unit of enzyme activity is defined as the change of 0.01 in A470 per minute, per mL of serum (plasma) in 1mL of reaction system.

b. Using 96-Well PlateUnit Definition: One unit of enzyme activity is defined as the change of 0.005 in A470 per minute, per mL of serum (plasma) in 1mL of reaction system.

Parameter Definitions:

• VTotal Reaction: Total reaction system volume, 0.245mL.
• VSample: Volume of sample added, 0.005mL.
• VTotal Sample: Volume of extraction solution added (for homogenization), 1mL.
• T: Reaction time, 1min.
• Cpr: Sample protein concentration, mg/mL.
• W: Sample mass, g.
• 500: Total number of bacteria or cells, 5 million.

Notes

• If the sample measurement value is less than 0.005, the reaction time can be extended to 3 to 5 minutes, and the final calculation should be divided by the extended reaction time.
• If the sample measurement value is greater than 0.5, the sample can be diluted with extraction buffer. Multiply the final calculated concentration by the corresponding dilution factor.

Gibson Assembly Protocol ​

1. Set up the following reaction on ice:

Recommended Amount of Fragments Used for Assembly

• Optimized cloning efficiency is 50–100 ng of vector with 2-3 fold molar excess of each insert. Use 5-fold molar excess of any insert(s) less than 200 bp. To achieve optimal assembly efficiency using 4-6 fragment assemblies, use a 1:1 molar ratio of each insert:vector. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%.
• Control reagents are provided for 5 experiments.
• If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.

2. Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at -20°C for subsequent transformation. Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
3. Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 µl of the assembly reaction, following the transformation protocol.

Ligation Protocol with T4 DNA Ligase (M0202)l ​

1. Set up the following reaction in a microcentrifuge tube on ice. (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.) Use NEBioCalculator to calculate molar ratios.

• The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.

2. Gently mix the reaction by pipetting up and down and microfuge briefly.
3. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
4. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
5. Heat inactivate at 65°C for 10 minutes.
6. Chill on ice and transform 1-5 µl of the reaction into 50 µl competent cells.

This is a quick-start protocol for purifying up to 10 µg of PCR products, ranging from 100 bp to 10 kb in size.

PCR Purification Kit ​

Notes Before Starting

• Add 96-100% ethanol to Buffer PE before use; check the bottle label for the correct volume.
• All centrifugation steps are performed at 17,900 x g (13,000 rpm) in a standard tabletop microcentrifuge at room temperature.
• Add a 1:250 volume of pH indicator I to Buffer PB.
• The yellow color of Buffer PB with pH indicator I indicates a pH of ≤7.5. This pH is crucial for the efficient adsorption of DNA to the membrane.
• If the purified DNA will be used for sensitive microarray applications, it may be better to use Buffer PB without the pH indicator.

Protocol

1. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix. If the mixture is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.0, and mix. The color will then turn yellow.
2. Place a QIAquick column in a 2 ml collection tube or a vacuum manifold.
3. To bind the DNA, apply the sample to the QIAquick column and centrifuge for 30-60 s or apply a vacuum until all the samples have passed through the column. Discard the flow-through and place the column back in the same tube.
4. To wash, add 750 µl of Buffer PE to the QIAquick column and centrifuge for 30-60 s or apply a vacuum. Discard the flow-through and return the column to the same tube.
5. Centrifuge the QIAquick column one more time for 1 minute to remove any residual wash buffer.
6. Place the QIAquick column in a clean 1.5 ml microcentrifuge tube. To elute the DNA, add 50 µl of Buffer EB (10 mM Tris-Cl, pH 8.5) or water (pH 7.0-8.5) to the center of the membrane and centrifuge for 1 minute. For a higher DNA concentration, add 30 µl of elution buffer, let the column stand for 1 minute, and then centrifuge.
7. If you plan to analyze the purified DNA on a gel, add 1 volume of Loading Dye to 5 volumes of the purified DNA. Mix the solution by pipetting up and down before loading the gel.

DNA Purification Kit ​

Packing List

Note: Add 39 ml of absolute ethanol to Solution II before first use.

Storage Conditions Store at room temperature, valid for one year.

Important Notes

• Before first use, add 39 ml of absolute ethanol to Solution II (Wash Buffer), mix well, and mark the bottle.
• Solution I is an irritant to the human body. Please be careful during operation and take appropriate protective measures to avoid direct contact or inhalation.
• All procedures in this kit should be performed at room temperature, without the need for an ice bath. All centrifugations should also be performed at room temperature.
• The waste collection tube is used multiple times during a single extraction. Do not discard it midway through the process.
• This product is for scientific research by professionals only. It must not be used for clinical diagnosis or treatment, nor for food or medicine. It should not be stored in a regular residence.
• For your safety and health, please wear a lab coat and disposable gloves during the procedure.

Operating Instructions

1. Add an equal volume of Solution I and mix well. For example, if the DNA sample volume is 100 µL, add 100 µL of Solution I. If the volume after mixing is large, you can add a portion of the sample to the purification column, centrifuge, and then add the remaining sample to continue the process. Mineral oil does not interfere with this kit.

2. Add the sample mixture (mixed with an equal volume of Solution I) to the DNA purification column and let it stand at room temperature for 1 minute. It is usually sufficient to wait a few seconds at room temperature until the solution has soaked into the column.

3. Centrifuge at maximum speed (16,000 x g, approximately 12,000-14,000 rpm) for 1 minute. Discard the liquid in the collection tube.

   • Note: It is crucial to reach 16,000 x g for this step, as a lower centrifugation speed will lead to reduced recovery efficiency.

4. Add 700 µL of Solution II to the DNA purification column and let it stand at room temperature for 1 minute.

5. Centrifuge at maximum speed for 1 minute to wash away impurities. Discard the liquid in the collection tube.

6. Add another 500 µL of Solution II and centrifuge at maximum speed for 1 minute to further wash away impurities. Discard the liquid in the collection tube.

7. Centrifuge at maximum speed again for 1 minute to remove residual liquid and allow any remaining ethanol to evaporate completely.

8. Place the DNA purification column on a new 1.5 mL microcentrifuge tube. Add 50 µL of Solution III to the center of the column membrane and let it stand for 1 minute.

   • Use a 1.5 mL microcentrifuge tube as the collection tube. Solution III should be added directly to the center of the column membrane to allow the liquid to be absorbed. If Solution III accidentally touches the tube wall, be sure to shake the tube to get the liquid to the bottom so that it can be absorbed by the column. Alternatively, you can use double-distilled water or Milli-Q grade ultrapure water instead of Solution III, but the pH of the water should be no less than 6.5. To obtain a higher concentration of DNA, you can add only 20-30 µL of Solution III, but the yield will be slightly reduced. Letting it stand for a longer time, such as 3-5 minutes, can help slightly improve the yield.

9. Centrifuge at maximum speed for 1 minute. The resulting liquid is the high-purity DNA.

Bacterial Glycerol Stock Preparation Protocol ​

Preparation of 50% Glycerol Solution

This procedure prepares 100 mL of sterile 50% glycerol solution, used to create a 25% final stock. Use a 250 mL flask for mixing.

1. Measure 50 mL of 100% Glycerol.
2. Measure 50 mL of Sterile ddH2O.
3. Combine the glycerol and ddH2O in the 250 mL flask. Mix well until the solution is homogeneous.
4. The solution was subjected to autoclaving for sterilization.
5. Allow the solution to cool completely to room temperature.

Preparation of Glycerol Stocks (in biological safety cabinet)

The following steps use the 50% glycerol solution to prepare 1 mL glycerol stocks with a 25% final glycerol concentration.

1. Aseptically pipette 500ul of the cooled, sterile 50% Glycerol Solution into each labeled vial.
2. Aseptically pipette 500ul of overnight bacterial culture into the same vial.
3. Gently mix the solution by inverting the capped tube 5-10 times to ensure even distribution of the glycerol and bacteria.
4. Immediately place the capped glycerol stocks into a −80∘C freezer for long-term storage.

Western Blot Protocol ​

With OriGene's Western Blot method, researchers can achieve reliable and accurate protein detection, making it simpler than ever to obtain consistent and optimal results.

1. Cracking (Lysis)

1. Pellet Bacteria: Take 1.5 ml of bacterial solution from each tube and centrifuge it at 8000 rpm for 1 min.
2. Remove Supernatant: Aspirate the supernatant as much as possible.
3. Prepare Lysis Buffer: The lysis buffers (A, B, and C) are added based on the bacterial pellet weight: 1 ml of buffer A, 1 µl of buffer B, and 10 µl of buffer C per 20 mg of bacterial pellet.
   • Calculation Example: Based on your W1(17.5 mg) and Control(14.5 mg) weights, the protocol suggests taking a total of 1011 µl of the pre-mixed lysis buffers (This total volume accounts for both samples and some redundancy, or assumes an approximate 20 mg average).
4. Add Lysis Buffer: Add the calculated amount of pre-mixed lysis buffer to each bacterial pellet (e.g., 885 µl for W1 and 733 µl for Control).
5. Resuspend and Shake: Resuspend the precipitate by vortexing. Then, shake it on a decolorization shaker at room temperature for 10 min.
6. Centrifuge: Centrifuge the lysate at 15000 rpm for 10 min at 4°C. (The centrifugation time may be extended if the precipitate is difficult to settle).
7. Collect Supernatant: Aspirate the supernatant and transfer it to a clean centrifuge tube for subsequent use.
   • Note: The gelatinous precipitate is difficult to remove and prone to accidental aspiration. Handle with care; it is not necessary to aspirate it completely clean.

2. BCA Assay for Protein Concentration

• 2.1. Standard Curve Preparation (Serial Dilution)

  1. Prepare 2 mg/ml Standard: Take 3.2 µl of the 25 mg/ml protein standard solution and add 36.8 µl of lysis buffer to prepare 40 µl of 2 mg/ml standard.
  2. Generate Lower Concentrations (Two-fold Dilutions):
     • 2 mg/ml Sample: Aspirate 20 µl into a new PCR tube.
     • 1 mg/ml: Add 20 µl of lysis buffer to the remaining 20 µl of the 2 mg/ml standard (halving the concentration). Aspirate 20 µl of this new 1 mg/ml solution.
     • Repeat: Repeat this step 3 more times to obtain standards of 0.5 mg/ml, 0.25 mg/ml, and 0.125 mg/ml.
  3. 0 mg/ml Standard (Blank): Pipette 20 µl of lysis buffer as the 0 mg/ml standard.

• 2.2. Sample Dilution and Assay

  1. Sample Dilution: Take 5 µl from each experimental sample and add 15 µl of lysis buffer for a 4-fold dilution.
  2. Prepare BCA Solution: Prepare the BCA assay solution at a ratio of Reagent A:Reagent B = 50:1. Calculate the total volume based on 200 µl × (6+number of samples), adding an appropriate amount of redundancy.
  3. Plate Setup:
     • Add 200 µl of the prepared BCA assay solution to each well of a 96-well plate.
     • Add 20 µl of the prepared sample dilution or protein standard solution to the appropriate wells.
  4. Incubation: Incubate the plate in a 37°C incubator for 20-30 minutes. Check the status multiple times; a clear distinction between the protein standards is acceptable.
  5. Reading and Calculation:
     • Measure the absorbance value at 562 nm using a microplate reader.
     • Draw a linear regression line with the protein standard solutions.
     • Calculate the concentration corresponding to the sample's absorbance value and multiply it by 4 to obtain the actual protein sample concentration.
     • Goal: The final protein concentration is preferably greater than 1 mg/ml.

3. PAGE Protein Electrophoresis

1. Sample Processing: Based on the calculated concentration from the BCA assay, dilute the more concentrated samples with lysis buffer to make the protein concentration consistent across all samples.
2. Add Sample Buffer: Add 5X sample buffer at a ratio of 1:4 (sample to 5X buffer).
3. Denaturation: Heat the prepared solutions at 95°C for 8 minutes for denaturation.
4. Loading:
   • Add 10 µl of the prepared solution to each loading well.
   • Marker: Add only 3 µl of the marker and dilute it to 10 µl with lysis buffer before loading.
5. Running Program: Run the gel with the following voltage/time sequence:
   • 60 V for 30 min
   • 90 V for 30 min
   • 120 V for 30 min
   • Stopping Point: Stop the run when the bottommost band is close to the bottom of the gel.

4. Transfer Membrane

1. Setup: The entire process must be carried out in the transfer buffer.
2. "Sandwich" Assembly: Assemble the transfer stack (sandwich) in the following order: Negative Electrode→Sponge→Filter Paper→Gel→Membrane→Filter Paper→Sponge→Positive Electrode
3. Transfer: Determine the transfer time and current based on the strip size. Run at 300 mA for 100 min.

5. Antibody Detection (Western Blot)

• 5.1. Initial Staining and Blocking

  1. Ponceau S Staining: Ensure the front side of the membrane faces up.
     • Stain with Ponceau S solution for 5-10 min.
     • Discard the solution, then add TBST for elution.
     • The presence of distinct red bands indicates successful protein transfer.
  2. Washing: Wash the membrane once with TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 5 min at room temperature.
  3. Blocking: Block non-specific binding on the membrane with freshly prepared 5% nonfat dried milk in TBST for 1 hour on a shaking platform at room temperature.

• 5.2. Antibody Incubation and Washing

  1. Primary Antibody: Incubate the membrane with a specific primary antibody diluted in TBST and 5% nonfat dried milk at the manufacturer's recommended dilution. Incubate with gentle agitation at 4°C overnight.
  2. Wash: Wash three times for 5 min each with TBST.
  3. Secondary Antibody: Incubate with OriGene's HRP-conjugated secondary antibody (TA130003) at a 1:20,000 dilution in TBST-5% nonfat dried milk for 1 hour at room temperature.
  4. Final Wash: Wash three times again for 5 minutes each with TBST.

• 5.3. Detection and Development

  1. Detection Solution: Use Western Blot Luminol Reagent and prepare it according to instructions.
  2. Incubation:
     • Lay the membrane on a plastic surface with the protein side up.
     • Add the mixed detection solution to the membrane.
     • Incubate for 1 minute.
     • Remove the excess solution.
  3. Exposure:
     • Place the wrapped blot, protein side up, in an imager.
     • Observe the result and take pictures.

Preparation and Transformation of Bacillus subtilis Competent Cells ​

This document provides a detailed protocol for preparing and transforming competent Bacillus subtilis cells.

1. Reagent and Strain Preparation

• You will need sterile ultrapure water, LB broth, LB agar plates, and optionally, 45% (v/v) sterile glycerol for long-term storage.
• To ensure high transformation efficiency, you must first activate a fresh culture of Bacillus subtilis. This involves streaking the bacteria onto an LB plate with the appropriate antibiotic and incubating overnight at 37°C.

2. Cultivation and Media Preparation

• Inoculate a single colony from the fresh plate into a test tube containing 3ml of LB broth with the appropriate antibiotic. Incubate overnight at 37°C with shaking (200 rpm) for 16-18 hours.
• The next morning, prepare Media I and Media II according to the provided tables using sterile ultrapure water and the specific preparation reagents (A, B, C, D, and E).

3. Preparing Competent Cells

• Step A: Transfer 80µl of the overnight culture to a 50ml sterile centrifuge tube containing 4ml of Media I. Incubate at 37°C with shaking (220 rpm) for 4-5 hours until the OD600 reaches 0.6-0.8.
• Step B: Add the 4ml culture to a 250ml sterile flask containing 36ml of Media II (1:9 ratio). Incubate at 37°C by shaking (100 rpm) for 90 minutes.
• Step C: Add 8µl of Reagent F and incubate for exactly 10 minutes at 37°C by shaking (100 rpm).
• Step D: Centrifuge at 4,000 × g for 3 minutes at 4°C to collect the cells. Resuspend the cells in 4ml of the remaining supernatant to obtain the competent cells.
• Step E: Dispense the competent cells into 200µl aliquots for a total of 20 tubes. For long-term storage, add 2ml of 45% sterile glycerol to the cell suspension before aliquoting into 200µl tubes (30 tubes total). Snap-freeze the tubes in liquid nitrogen and store at -80°C.

4. Transformation

• Thaw the competent cells on ice.
• Add 0.5-5µg of plasmid to each tube and mix gently.
• Incubate at 37°C by shaking (200 rpm) for 90-120 minutes.
• Centrifuge at 4,000 × g for 1 minute to collect the cells.
• Resuspend the pellet in 50µl of the remaining supernatant and spread onto an LB plate with the corresponding antibiotic.
• Incubate the plate overnight at 37°C.

Prepare an agarose gel ​

1. Gel Apparatus Assembly

• Place the gel tray on a level workbench. Assemble the gel base plate and insert the sample comb.

2. Selecting Gel Concentration

• Choose the appropriate gel concentration based on the size of the DNA sample you need to run. As per the provided chart, the recommended concentration for a DNA sample size of 800-5000kb is a 1% agarose gel.

3. Agarose Gel Preparation

• Weigh the required amount of agarose powder according to your desired gel concentration and add it to an Erlenmeyer flask. Add the corresponding volume of 1x TAE or 1x TBE buffer. Heat the mixture in a microwave oven until the agarose powder is completely dissolved. For a 1% gel, add 1g of agarose powder to 100ml of 1x buffer solution.

4. Gel Casting

• Once the agarose solution has cooled to 45-60°C, pour it into a beaker. Add a nucleic acid dye, mix well, then pour the solution into the gel mold and insert the comb. Allow it to solidify at room temperature for 20-30 minutes to form a uniform gel with well-defined sample wells.

FastPure® Cell/Tissue Total RNA Isolation Kit V2-RC112 (Bacterial Extraction Protocol) ​

I. Sample Processing

The protocol outlines different methods based on the bacterial type: Gram-Negative Bacteria, Gram-Positive Bacteria, and Complex Bacteria (Gram-positive with particularly thick cell walls).

Gram-Negative Bacteria

1. Take 1-5 ml of bacterial culture (<1.0×10⁹ bacteria) and centrifuge at 12,000 rpm (~13,400×g) for 2 minutes, then discard the supernatant (culture medium).
   • Note: Bacterial count can be measured by a spectrophotometer; OD600=1.0 is approximately 1.5×10⁹ bacteria/ml.
2. Add 100 µl of Lysozyme (10 mg/ml) (Vazyme #DE103) and vortex to fully resuspend the pellet.
3. Add 500 µl of Buffer RL and vortex until no visible bacterial clumps remain.
4. Proceed to "II. RNA Extraction".

Gram-Positive Bacteria

1. Take 1-5 ml of bacterial culture (<1.0×10⁹ bacteria) and centrifuge at 12,000 rpm (~13,400×g) for 2 minutes, then discard the supernatant.
   • Note: OD600=1.0 is approximately 1.5×10⁹ bacteria/ml.
2. Add 1 ml of 70% ethanol (prepared with RNase-free H₂O), vortex to resuspend, incubate on ice for 20 minutes, centrifuge at 12,000 rpm (~13,400×g) for 1 minute, and discard the supernatant.
3. Add 100 µl of Lysozyme (30 mg/ml) (Vazyme #DE103), vortex to resuspend, and incubate at 37°C for 15 minutes.
   • Note: For some bacteria with thicker cell walls (e.g., Staphylococcus aureus), the incubation time can be extended to 1-2 hours to aid cell wall lysis. Adjust the incubation time as appropriate for different bacteria.
4. Add 500 µl of Buffer RL and vortex until no visible bacterial clumps remain.
5. Proceed to "II. RNA Extraction".

Complex Bacteria

1. Take 1-5 ml of bacterial culture (<1.0×10⁹ bacteria) and centrifuge at 12,000 rpm (~13,400×g) for 2 minutes, then discard the supernatant.
   • Note: OD600=1.0 is approximately 1.5×10⁹ bacteria/ml.
2. Add 1 ml of 70% ethanol (prepared with RNase-free H₂O), vortex to resuspend, incubate on ice for 20 minutes, centrifuge at 12,000 rpm (~13,400×g) for 1 minute, and discard the supernatant.
3. Add 100 µl of Lysozyme (30 mg/ml) (Vazyme #DE103) and incubate at 37°C for 15 minutes.
4. Add 500 µl of Buffer RL and vortex until no visible bacterial clumps remain.
5. Transfer the mixture to a safe-lock tube containing 400 mg of glass beads (150-500 μm).
6. Vortex at maximum speed for 10 minutes.
7. Centrifuge at 12,000 rpm (~13,400×g) for 30 seconds and keep the supernatant.
8. Proceed to "II. RNA Extraction".

II. RNA Extraction

1. Transfer the lysed sample to a FastPure gDNA-Filter Columns III (placed in a collection tube) and centrifuge at 12,000 rpm (~13,400×g) for 30 seconds. Discard the column and collect the filtrate.
2. Add 0.5× filtrate volume of absolute ethanol to the filtrate (for liver tissue, add 1× filtrate volume of 50% ethanol) and mix thoroughly.
   • Note: Turbidity or flocculent precipitate after adding ethanol is normal; mix well and proceed to the next step.
3. Transfer the entire mixture from Step 2 to a FastPure RNA Columns III (placed in a collection tube) and centrifuge at 12,000 rpm (~13,400×g) for 30 seconds. Discard the flow-through.
4. Add 700 µl of Buffer RW1 to the column and centrifuge at 12,000 rpm (~13,400×g) for 30 seconds. Discard the flow-through.
5. Add 700 µl of Buffer RW2 (with absolute ethanol added) to the column and centrifuge at 12,000 rpm (~13,400×g) for 30 seconds. Discard the flow-through.
6. Add 500 µl of Buffer RW2 (with absolute ethanol added) to the column and centrifuge at 12,000 rpm (~13,400×g) for 2 minutes. Carefully remove the column from the collection tube, avoiding contact with the flow-through to prevent contamination.
7. (Optional): If there is liquid residue on the column or if it contacted the flow-through, discard the flow-through, place the FastPure RNA Columns III back in the collection tube, and centrifuge empty at 12,000 rpm (~13,400×g) for 1 minute (dry spin) to prevent ethanol contamination.
8. Carefully transfer the column to a new RNase-free Collection Tube (1.5 ml).
9. Add 50-200 µl of RNase-free ddH₂O to the center of the column membrane, let it sit at room temperature for 1 minute, and centrifuge at 12,000 rpm (~13,400×g) for 1 minute to elute the RNA.
   • Note: To increase yield, the RNase-free ddH₂O can be pre-heated to 65°C and allowed to sit for 2-5 minutes after adding to the membrane, or a second elution can be performed after the first centrifugation.
10. The extracted Total RNA can be used directly for downstream experiments or stored at -85°C to -65°C.

PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) Reverse Transcription Reaction: Probe qPCR Analysis Method ​

1. Genomic DNA Elimination Reaction (gDNA Eraser Reaction) The reaction mixture components are prepared on ice. To ensure accurate reaction preparation, first prepare a Master Mix for (Number of Reactions +2), then aliquot to each reaction tube, and finally add the RNA sample.

2. Reverse Transcription Reaction (RT Reaction) The reaction solution should be prepared on ice. To ensure accurate preparation, first prepare a Master Mix for (Number of Reactions +2), then aliquot 10 µl to each reaction tube *3. Gently mix and immediately start the reverse transcription reaction.

• 1. For the 20 µl reverse transcription reaction system, a maximum of 2 µg of Total RNA can be used for the Probe qPCR analysis method.
• 2. When reacting at room temperature, the incubation time can be extended up to 30 minutes.
• 3. If not using a Master Mix, when adding reagents to the Step 1 solution, first add the RNase Free ddH₂O and 5× PrimeScript Buffer 2 (for Real Time) and mix well to fully inhibit the gDNA Eraser activity. Then add the RT Primer Mix and PrimeScript RT Enzyme Mix I, gently mix, and proceed with the reverse transcription reaction.
• 4. Using the RT Primer Mix enables efficient cDNA synthesis. For different experimental purposes, you can choose not to use the RT Primer Mix and instead select Oligo dT Primer or Gene Specific Primer (GSP) for the reverse transcription reaction. The primer usage amounts are as follows:
  • Oligo dT Primer: 50 pmol/20 µl reaction system
  • Gene Specific Primer: 5 pmol/20 µl reaction system
• 5. The reverse transcription system volume can be scaled up as needed.
• 6. When using Gene Specific Primer, it is recommended to set the reverse transcription reaction condition to 42°C for 15 minutes. If non-specific amplification occurs in the PCR reaction, increasing the temperature to 50°C may offer improvement.
• 7. If the synthesized cDNA needs to be stored long-term, please store it at -20°C or lower.

Note:

1. In the reverse transcription reaction, the amount of RT Primer Mix used is 1 µl for the TB Green qPCR method, and 4 µl for the Probe qPCR analysis method.
2. The resulting RT reaction solution (cDNA) added to the subsequent Real Time PCR reaction system should not exceed 1/10 (V/V) of the total Real Time PCR reaction volume.