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Preparation of basic experimental material
Molecular Biology Experiment
Microbiology Experiment
Plant Experiment
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Preparation of Basic Experimental Material

1. LB

Materials:

  • Tryptone
  • Yeast extract
  • NaCl AR
  • dH2O
  • Conical flask
  • Microwave
  • Magnetic stirrer and rotor

Protocol:

  1. Weigh reagents in the following proportions:
    Substance Concentration
    Tryptone 10 g/L
    Yeast extract 5 g/L
    NaCl 10 g/L
  2. Deposit them all into the flask.
  3. Add distilled water to the expected volume.
  4. Stir the mix until the powders are completely dissolved.
  5. Autoclave for 20 minutes at 121°C.
  6. Store at 4°C.

2. LB Agar

Materials:

  • Tryptone
  • Yeast extract
  • NaCl AR
  • Agar
  • dH2O
  • Conical flask
  • Microwave
  • Magnetic stirrer and rotor

Protocol:

  1. Weigh reagents in the following proportions:
    Substance Concentration
    Tryptone 10 g/L
    Yeast extract 5 g/L
    NaCl 10 g/L
  2. Deposit them all into the flask.
  3. Add distilled water to the expected volume.
  4. Stir the mix until the powders are completely dissolved.
  5. Add 1.5% weight agar into the flask.
  6. Autoclave for 20 minutes at 121°C.
  7. Store at 4°C.

3. Preparation of LB-Antibiotics

  • If the medium does not contain agar: Add one thousandth of a volume of antibiotic solution, mix thoroughly.
  • If the medium does contain agar: When the temperature is reduced to approximately 45°C, add one thousandth of a volume of antibiotic solution, then mix it thoroughly. After antibiotics are added, it should not be heated again.

4. Preparation of Agarose Gel

Materials:

  • Agarose
  • 1x TAE
  • Graduated cylinder
  • Conical flask
  • Pipettor and pipette
  • Microwave
  • 10,000x nucleic acid dye

Protocol:

  1. Add the exact weight of agarose to the measuring cylinder to ensure the correct concentration.
  2. Add 1x TAE of the required volume.
  3. Heat the solution in the microwave until it boils.
  4. Take the cylinder out and let it cool down until it can be touched by hands.
  5. Add nucleic acid dye that is 1/10,000 of the total volume of the system.
  6. Pour into electrophoresis box, place the comb, and wait until it solidifies.
  7. Take out the comb carefully.

5. TY Medium

Materials:

  • Tryptone
  • Yeast extract
  • CaCl2 AR
  • dH2O
  • Conical flask
  • Microwave
  • Magnetic stirrer and rotor

Protocol:

Substance Concentration
Tryptone 5 g/L
Yeast extract 3 g/L
CaCl2 0.6 g/L
  1. Deposit them all into the flask.
  2. Add distilled water to the expected volume.
  3. Stir the mix until the powders are completely dissolved.
  4. Autoclave for 20 minutes at 121°C.
  5. Store at 4°C.

6. TY Agar

Materials:

  • Tryptone
  • Yeast extract
  • CaCl2 AR
  • Agar
  • dH2O
  • Conical flask
  • Microwave
  • Magnetic stirrer and rotor

Protocol:

Substance Concentration
Tryptone 5 g/L
Yeast extract 3 g/L
CaCl2 0.6 g/L
  1. Deposit them all into the flask.
  2. Add distilled water to the expected volume.
  3. Stir the mix until the powders are completely dissolved.
  4. Add 2% weight agar into the flask.
  5. Autoclave for 20 minutes at 121°C.
  6. Store at 4°C.

If needed, 5% sucrose can be added to TY or TY agar.

7. 5xM9 Medium

Materials:

  • Na2HPO4
  • KH2PO4
  • NaCl AR
  • CaCl2 AR
  • MgSO4·7H2O
  • NH4Cl
  • Glucose
  • dH2O
  • Conical flask
  • Microwave
  • Magnetic stirrer and rotor

Protocol:

  1. Weigh reagents in the following proportions for the 5xM9 basic salt solution:
    Substance Concentration
    Na2HPO4 33.9 g/L
    KH2PO4 15.0 g/L
    NaCl 2.5 g/L
  2. Heat to dissolve, make up to 400 ml, high-temperature sterilize, and separately prepare the following three solvents.
  3. Weigh reagents in the following proportions for 1M MgSO4·7H2O:
    Substance Concentration
    MgSO4·7H2O 246.5 g/L
  4. Weigh reagents in the following proportions for 1M CaCl2:
    Substance Concentration
    CaCl2 111 g/L
  5. Weigh reagents in the following proportions for 100xNH4Cl:
    Substance Concentration
    NH4Cl 100 g/L
  6. Prepare the three solvents separately and high-temperature sterilize.
  7. Weigh reagents in the following proportions for 20% Glucose Solution:
    Substance Concentration
    Glucose 200 g/L
  8. Sterilize by filter membrane.
  9. Take 100 ml of 5xM9 basic salt solution, add 10 ml of 20% Glucose Solution, 1 ml of 1M MgSO4·7H2O, and 50 µL of CaCl2 solution, and make up to 500 ml.
  10. Based on the concentration gradient set, add the corresponding amount of 100xNH4Cl as required.

Molecular Biology Experiment

1. Genome Extraction

Materials:

  1. Centrifuge tubes (1.5 mL & 2.0 mL)
  2. G+ Bacteria Genomic DNA Kit (Zomanbio) (Lysozyme (100 mg/mL), cell suspension solution, Buffer A, lysis solution S, Buffer B, Buffer C, Washing Solution W2, Elution Buffer TE, Elution Buffer Columns, Collection Tube)
  3. RNase A (10 mg/mL) (Zomanbio)
  4. ddH2O
  5. Ethanol
  6. Centrifugal machine
  7. Vortex machine
  8. Water bath

Protocol:

  1. Centrifugate 1-5 mL bacteria solution for 1 min at 13,400xg to retain the pellet.
  2. Add 500 μl cell suspension solution (with Lysozyme added) to the bacterial pellet, vortex until the pellet is re-suspended. Incubate at 37℃ for 30 minutes, inverting every 5-10 minutes. Centrifuge at 13,400xg for 2 minutes and thoroughly remove the supernatant.
  3. Add 225 μl Buffer A and mix thoroughly.
  4. Add 6 μl RNase A (10 mg/mL) solution, vortex for 5 seconds and mix well.
  5. Add 25 μl lysis solution S and mix thoroughly. Incubate at 56℃ for 20 minutes, inverting 1-2 times during this period.
  6. Add 250 μl Buffer B, vortex for 5 seconds and mix thoroughly.
  7. Add 250 μl ethanol, vortex for 15 seconds. Transfer the mixture to the column and place the column in the collection tube.
  8. Centrifuge at 13,400xg for 30 seconds, discard the supernatant. Place the absorption column in the collection tube.
  9. Add 500 μl Buffer C, centrifuge at 13,400xg for 30 seconds, discard the supernatant, and place the column back into the collection tube.
  10. Add 600 μl Washing Solution W2 (with ethanol added), centrifuge at 13,400xg for 30 seconds, discard the supernatant, and place the column back into the collection tube.
  11. Add an additional 500 μl Washing Solution W2 (with ethanol added), centrifuge at 13,400xg for 30 seconds, discard the supernatant, and place the column back into the collection tube.
  12. Centrifuge for 2 minutes at 13,400xg. Open the column and let it sit at room temperature for a few minutes.
  13. Place the column in a new 1.5 mL centrifuge tube, add 100-200 μl elution buffer TE, and let it sit at room temperature for 2-5 minutes.
  14. Centrifuge for 2 minutes at 13,400xg. Transfer the liquid in the collection tube to the column.
  15. Dispose of the column and store the eluate at -20℃.

2. PCR

2.1 General PCR

Goal: Amplify a gene or add homology arms to a sequence.

Materials:

  • PCR tubes (200 μl)
  • GoldenStar T6 Super PCR Mix (1.1x)
  • Forward primer (10 μM)
  • Reverse primer (10 μM)
  • Template
  • Thermocycler

Protocol:

  • Add in a PCR tube:
    Substance Volume (system 25μl/50μl)
    Golden Mix (23-x) μl/(46-x) μl
    Forward primer (10 μM) 1 μl/2 μl
    Reverse primer (10 μM) 1 μl/2 μl
    Template x μl (depends)
  • Place the tube in the thermocycler and start corresponding program.
  • After the sample temperature decreases to 4℃, store the result at -20℃.

    • 2.2 Fusion PCR

    Goal: Connect two DNA fragments containing homologous arms together.

    Materials:

    1. PCR tubes (200 μl)
    2. GoldenStar T6 Super PCR Mix (1.1x)
    3. Template (containing homologous arms)
    4. Forward primer (10 μM)
    5. Reverse primer (10 μM)
    6. Thermocycler

    Protocol:

  • Add in a PCR tube:
    Substance Volume (system 25μl/50μl)
    Golden Mix (23-x-y) μl/(46-x-y) μl
    Template1 x μl (depends)
    Template2 y μl (depends)
  • Add 1 μl/2 μl of each of the two primers.
  • Place the tube in the thermocycler and start corresponding program.
  • After the sample temperature decreases to 4℃, store the result at -20℃.

    • 2.3 Colony PCR

    Goal: To determine whether a certain sequence exists in the thallus.

    Materials:

    1. PCR tubes (200 μl)
    2. 2 x Taq Master Mix (dye) / 2 x T5 Super PCR Mix (Basic)
    3. Forward primer (10 μM)
    4. Reverse primer (10 μM)
    5. ddH2O
    6. Thermocycler

    Protocol:

    1. Add in a PCR tube:
      Substance Volume (system 25μl)
      2 x Taq Master Mix (dye) / 2 x T5 Super PCR Mix (Basic) 12.5 μl
      Forward primer (10 μM) 1 μl
      Reverse primer (10 μM) 1 μl
      ddH2O 10.5 μl
    2. Usually, we configure more premix than the required number of systems in a 1.5 mL centrifuge tube, then separate them into PCR tubes.
    3. Place the tube in the thermocycler and start corresponding program.
    4. After the sample temperature decreases to 4℃, stop the program and use the results for AGE detection.

    Plasmid Extraction

    3.1 Zomanbio

    Materials:

    • Centrifuge tubes (1.5 mL & 2.0 mL)
    • Fast Plasmid Miniprep Kit (Zomanbio) (RNase A (10 mg/mL), Solution K1, Solution K2, Solution K3, Deproteinized solution W1, Buffer V2, Washing Solution W2, Elution Buffer TE, Elution Buffer Columns, Collection Tube)
    • Centrifugal machine
    • Vortex machine

    Protocol:

    1. Centrifugate 1-5 mL bacteria solution 1 min at 13,400 x g, retain precipitation.
    2. Add 250 μl Buffer K1 (RNase added) in the bacteria precipitation, vortex it until the thallus is resuspended.
    3. Add 250 μl buffer K2, gently mix it until the liquid looks clear.
    4. Add 350 μl buffer K3, immediately mix it gently.
    5. Centrifugate the tube 2 min at 13,400xg, transfer the supernatant to column and put it in the collection tube, centrifugate 30-60 s at 13,400xg. Discard supernatant, replace the column into the collection tube.
    6. If the bacteria is E. coli BL21 (DE3) or Pseudomonas fluorescens 2P24, add 500 μl Deproteinized solution W1 in column and centrifugate 30-60 s at 13,400xg. If the bacteria is E. coli DH5 α, skip this step.
    7. Add 500-600 μl Washing Solution W2 (ethanol added) in column and centrifugate 20 s at 13,400xg. Repeat this step.
    8. Centrifugate 1 min at 13,400xg.
    9. Put the column in a new centrifuge tube, add 80-150 μl elution buffer TE. Place it for 1 min.
    10. Centrifugate 1 min at 13,400xg. Discard the column and store the result at -20℃.

    3.2 TransGen

    Materials:

    • Centrifuge tubes (1.5 mL & 2.0 mL)
    • EasyPure Plasmid Miniprep Kit (TransGen) (RNase A (10 mg/mL), Resuspension Buffer (RB), Lysis Buffer (LB), Neutralization Buffer (NB), Wash Buffer (WB), Elution Buffer (EB), Mini-Plasmid Spin Column with Collection Tubes)
    • Centrifugal machine
    • Vortex machine

    Protocol:

    1. Before use, RNase A was added to RB and stored at 2-8℃. Add different volumes of 100% ethanol to WB.
      2. LB Media Volume RB (μl) LB (μl) NB (μl)
      5 mL 250 250 350
      5-10 mL 500 500 700
      10-15 mL 750 750 1,050
      15-20 mL 1,000 1,000 1,400
    2. Take the overnight culture, centrifuge at 10,000xg for 1 minute, and remove the supernatant.
    3. According to the table, add colorless solution RB (containing RNase A), vortex to resuspend the bacterial pellet, ensuring no small clumps remain.
    4. According to the table, add blue solution LB, gently invert 4-6 times to mix, ensuring complete lysis of the bacteria, forming a blue and clear solution. The color change from semi-transparent to clear blue indicates complete lysis.
    5. According to the table, add yellow solution NB, gently mix 5-6 times (the color change from blue to yellow indicates complete mixing and neutralization), until a compact yellow precipitate forms, then let it stand at room temperature for 2 min.
    6. Centrifuge at 12,000xg for 5 min, carefully transfer the supernatant to a centrifuge column. Centrifuge at 12,000xg for 1 min, discard the flow-through.
    7. Add 650 μl solution WB, centrifuge at 12,000xg for 1 min, and discard the flow-through.
    8. Centrifuge at 12,000xg for 1-2 min to thoroughly remove residual WB.
    9. Place the centrifuge column in a clean centrifuge tube, add 20 μl EB to the center of the column, and let it stand at room temperature for 1 min. (Pre-warming EB in a 60-70°C water bath improves the outcome).
    10. Centrifuge at 10,000xg for 1 min to elute the DNA, and store the eluted DNA at -20°C.

    3.3 Mei5bio

    Materials:

    • Centrifuge tubes (1.5 mL & 2.0 mL)
    • M5 HiPer Multi-color Plasmid Miniprep Kit (Mei5bio) (RNase A (10 mg/mL), Solution Ⅰ, Solution Ⅱ, Solution Ⅲ, Buffer WB1 (with anhydrous ethanol added), Buffer WB2 (with anhydrous ethanol added), Buffer BL, Buffer EB, Washing Solution W2, Elution Buffer TE, Elution Buffer Columns, Collection Tube)
    • Centrifugal machine
    • Vortex machine

    Protocol:

    1. Column Equilibration: Add 400 μL of Buffer BL to the centrifugal adsorption column, let it stand for 1 minute, centrifuge at 12,000 rpm at room temperature for 1 minute, discard the waste liquid in the collection tube, and place the centrifugal adsorption column back into the collection tube.
    2. Take 1-5 mL of overnight culture, centrifuge at 12,000 rpm at room temperature for 1 minute, and remove the supernatant as completely as possible.
    3. Add 250 μL of Solution I, vortex or pipette thoroughly to resuspend the bacteria evenly, resulting in a uniformly turbid brown-red color.
    4. Add 250 μL of Solution II, gently invert to mix well, allowing the bacteria to fully lyse until the solution becomes clear and viscous purple-red.
    5. Add 350 μL of Solution III, immediately and gently invert to mix, a precipitate with red and yellow layers will be visible, continue mixing until it turns completely yellow, let it stand at room temperature for 2 min, then centrifuge at 12,000 rpm for 5 min.
    6. Carefully transfer the supernatant to the centrifugal adsorption column, let it stand for 2 min to allow the plasmid to bind fully with the silica membrane of the column. Centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate in the collection tube.
    7. Add 500 μL of Buffer WB2, centrifuge at 12,000 rpm at room temperature for 30 seconds, and discard the filtrate in the collection tube. Repeat this step once.
    8. Centrifuge at 12,000 rpm at room temperature for 2 min to dry the residual liquid.
    9. Place the centrifugal adsorption column in a new 1.5 mL plastic centrifuge tube (provided by the user), add 50-100 μL of elution buffer EB, let it stand at room temperature for 2 min.
    10. Centrifuge at 12,000 rpm for 1 minute, and the solution at the bottom of the centrifuge tube is the plasmid DNA. Store the eluted DNA at -20℃.

    Plasmid Digestion

    Goal: To cleave a vector using restriction enzymes to prepare them for ligation.

    4.1 pBBR1MCS-2, EcoRⅠ + XmaⅠ

    Materials:

    • EcoRⅠ
    • XmaⅠ
    • pBBR1MCS-2
    • rCutSmart Buffer (10x)
    • ddH2O
    • 200 μL centrifuge tube
    • Water bath at 37℃
    • Water bath at 65℃

    Protocol:

    Substance Volume
    EcoRⅠ 0.1 μl
    XmaⅠ 0.1 μl
    pBBR1MCS-2 x μl (depends)
    rCutSmart Buffer (10x) 1 μl
    ddH2O (8.8-x) μl
    1. Keep warm in a water bath at 37℃ for 1 hour.
    2. Keep warm in a water bath at 65℃ for 20 minutes.
    3. Correct bands were extracted after electrophoresis.

    4.2 pJQ200SK, SmaⅠ

    Materials:

    • SmaⅠ
    • pJQ200SK
    • 10X QuickCut Green Buffer
    • ddH2O
    • 200 μL centrifuge tube
    • Water bath at 37℃
    • Water bath at 80℃

    Protocol:

    Substance Volume
    SmaⅠ 1 μl
    pJQ200SK x μl (depends)
    10X QuickCut Green Buffer 1 μl
    ddH2O (8-x) μl
    1. Keep warm in a water bath at 37℃ for 1 hour.
    2. Keep warm in a water bath at 80℃ for 15 minutes.
    3. Correct bands were extracted after electrophoresis.

    Agarose Gel Electrophoresis (AGE)

    Goal: Separating DNA bands according to their length, and estimating the length and DNA concentration of each band. When digested products or recombinant products are subjected to electrophoresis, care should be taken to use the original plasmids for control.

    Materials:

    • Ready-made agarose gel
    • DNA sample
    • 10 x loading buffer (dye)
    • DNA Ladder/Marker
    • 1 x TAE
    • Electrophoresis tank

    Protocol:

    1. Place the gel on the gel tray within the electrophoresis system, make sure the wells are located at the negative electrode.
    2. Mix loading buffer with the DNA sample if the sample is not coloured. Usually, the loading buffer has one tenth the volume of the sample, but the volume should not be less than 0.3 μl.
    3. Load either the DNA ladder or the samples within the wells. Make sure that the sample or ladder sinks to the bottom of the well. The volume should be between 3 μl to 5 μl, depending on the well's capacity.
    4. Run the gel for 20-40 minutes, depending on the situation. Ensure the voltage is set to 100-150 V.

    DNA Extraction from Agarose Gel

    Materials:

    • Centrifuge tubes (1.5 mL)
    • EasyPure Quick Gel Extraction Kit (TransGen) (Wash Buffer WB, Elution Buffer EB, Gel Solubilization Buffer GSB, Gel Spin Columns with Collection Tubes)
    • ddH2O
    • Centrifugal machine
    • Vortex machine
    • Water bath

    Protocol:

    1. Carefully cut the target band on agarose gel and put it in a centrifuge tube.
    2. Add 600 μl GSB. Heat it at 55℃ using a water bath for 6-10 minutes. Mix intermittently to ensure the gel is completely melted.
    3. After the gel dissolves, allow the solution to cool to room temperature, transfer the liquid to a column, and put it in the collection tube. Centrifuge for 60 seconds at 10,000 xg. Discard the flow-through liquid.
    4. Add 650 μl WB, centrifuge for 60 seconds at 10,000 xg. Discard the flow-through liquid.
    5. Centrifuge for 1-2 minutes at 10,000 xg to completely remove residual WB.
    6. Place the centrifuge column in a clean centrifuge tube, open the cap and let it stand for 1 minute to allow the residual ethanol to evaporate completely. Then add 20 μl of deionized water (pre-warmed) to the center of the column and let it stand at room temperature for 1 minute.
    7. Centrifuge for 60 seconds at 10,000 xg. Discard the column and store the result at -20℃.

    Gibson Assembly

    Goal: To assemble the different DNA parts retrieved by PCR.

    Materials:

    • PCR tubes (200 μl)
    • ClonExpress Ultra One Step Cloning Kit (2xClonExpress Mix, pCE-Zero vector linearized (50 ng/μl), 500bp control insert (20 ng/μl))
    • ddH2O
    • Thermocycler

    Protocol:

    1. Calculate the required amount of DNA for the recombination reaction according to the formula:
      • Single-fragment: Optimal cloning vector usage = [0.02 x number of base pairs of cloning vector] ng (0.03 pmol) Optimal insert fragment usage = [0.04 x number of base pairs of insert fragment] ng (0.06 pmol)
      • Multi-fragment: Optimal cloning vector usage = [0.02 x number of base pairs of cloning vector] ng (0.03 pmol) Optimal usage for each fragment = [0.02 x number of base pairs of each fragment] ng (0.03 pmol)
    2. Prepare the following reaction system on ice:
      Components Recombination Reaction Negative Control-1 Negative Control-2 Positive Control
      Linearized Vector X μl X μl 0 μl 1 μl
      n Insert Fragments (n≤5) Y1+Y2...+Yn μl 0 μl Y1+Y2...+Yn μl 1 μl
      2x ClonExpress Mix 5 μl 0 μl 0 μl 5 μl
      ddH2O to 10 μl to 10 μl to 10 μl to 10 μl
    3. Gently mix well with a pipette (do not vortex mix), briefly centrifuge to collect the reaction liquid at the bottom of the tube.
    4. Single-fragment recombination reaction, 50°C, 5 min; cool to 4°C or immediately place on ice.
    5. Multi-fragment recombination reaction, 50°C, 15 min; cool to 4°C or immediately place on ice.

    RNA Extraction

    Materials:

    • StarSpin Plant RNA Kit (Buffer RPA, RNase-free DNase, Buffer DB, Buffer RW1, Buffer RW2, Buffer TB, Filtration Columns with Collection Tubes-RF, Spin Columns with Collection Tubes-RC, 1.5ml Centrifuge Tubes (DNase/RNase-free))

    Protocol:

    1. Take 500 μl of Buffer RPA (which has been supplemented with β-mercaptoethanol) and add it to a 1.5 ml RNase-free centrifuge tube. After grinding the tissue sample in liquid nitrogen, add the powdered sample (50-100 mg) to the 1.5ml centrifuge tube containing 500 μl of Buffer RPA. Vortex vigorously to mix well until there are no visible precipitates in the lysis solution.
    2. Centrifuge at 12,000rpm for 2 minutes.
    3. Place the filtration column into a collection tube, then transfer the supernatant collected from the previous centrifugation step into the Filtration Columns with Collection Tubes-RF (with the filtration column placed in the collection tube), centrifuge at 12,000 rpm for 2 minutes. Carefully aspirate the filtrate from the collection tube into a new 1.5 ml RNase-free centrifuge tube, avoiding contact with the cell debris precipitate in the collection tube.
    4. Slowly add anhydrous ethanol equivalent to 0.4 times the volume of the filtrate. Mix well (a precipitate may form at this point), and transfer the resulting clear liquid and precipitate together into the 5pin Columns with Collection Tubes-RC (with the adsorption column placed in the collection tube), centrifuge at 12,000 rpm for 15 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
    5. Add 350 μl of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
    6. Prepare the RNase-free DNase working solution: take 10 μl of RNase-free DNase and add it to a new RNase-free centrifuge tube, add 70 μl of Buffer DB, and mix well to prepare an RNase-free DNase working solution with a final volume of 80 μl.
    7. Apply 80 μl of the RNase-free DNase working solution to the center of the adsorption column and let it stand at room temperature for 15 minutes.
    8. Add 350 μl of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
    9. Add 500 μl of Buffer RW2 (which has been supplemented with anhydrous ethanol) to the adsorption column, let it stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute. Discard the waste liquid in the collection tube and place the adsorption column back into the collection tube.
    10. Repeat step 9 once.
    11. Place the adsorption column back into the collection tube and centrifuge at room temperature at 12,000 rpm for 3 minutes.
    12. Place the adsorption column into a new 1.5ml centrifuge tube (DNase/RNase-free), add 50-100 μl of Buffer TB, let it stand at room temperature for 1-2 minutes, and centrifuge at 12,000 rpm for 1 minute to obtain the RNA solution. The RNA obtained should be used immediately or aliquoted and stored at -80℃ to avoid repeated freeze-thaw cycles.

    Reverse Transcription

    Goal: Reverse extracted RNA into cDNA.

    Materials:

    • RNA template
    • StarScript III All-in-one RT Mix with gDNA Remover Kit (StarScript III All-in-one RT Mix; 5×StarScript III All-in-one RT Buffer; Nuclease-free Water (DEPC-treated))
    • 1.5ml Centrifuge Tubes (DNase/RNase-free)

    Protocol:

    Substance Volume
    RNA template ≤ 1 μg total RNA or ≤ 0.1 μg poly(A) mRNA
    StarScript III All-in-one RT Mix 1 μl
    5×StarScript III All-in-one RT Buffer 4 μl
    Nuclease-free Water (DEPC-treated) up to 20 μl
    • React under the following conditions:
      Temperature Time
      37℃ 2 min
      50℃ 15 min
      80℃ 2 min
    • The cDNA obtained after the reaction is completed is placed on ice for subsequent experiments or frozen storage.

    qPCR

    Goal: Detecting the expression of target genes at the level of conversion rate.

    Materials:

    • DNA template
    • Forward primer
    • Reverse primer
    • 2×RealStar Fast SYBR qPCR Mix (UNG, Low DNA) Kit (2×RealStar Fast SYBR qPCR Mix (UNG, Low DNA); High/Low ROX Reference Dye)
    • Centrifuge Tubes
    • Sterile Water

    Protocol:

    Substance Volume
    DNA template 1 μl
    Forward primer 0.5 μl
    Reverse primer 0.5 μl
    2×RealStar Fast SYBR qPCR Mix (UNG, Low DNA) 10 μl
    High/Low ROX Reference Dye 0.4 μl
    Sterile Water up to 20 μl
    • React under the following conditions in real-time instrument:
      Temperature Time Cycles
      50℃ 5 min  
      95℃ 2 min  
      95℃ 10 s 40
      60℃ 30 s  

    Microbiology Experiment

    1. Transformation of chemically competent E. coli

    Materials:

    • LB medium
    • LB agar plates with antibiotics (depends)
    • Crushed ice
    • Plasmid used to transform
    • Chemically competent cell
    • Centrifuge tube (1.5 mL)

    Protocol:

    1. Add 100 μl chemically competent cell and 10 μl of plasmid in the tube, mix gently and softly.
    2. Incubate the tube for 30 minutes on ice.
    3. Incubate for 90 seconds at 42°C, then immediately place on ice for 2 minutes.
    4. Add 700-900 μl (depends) of LB liquid medium.
    5. Incubate for 45 minutes under agitation at 200 rpm.
    6. Centrifuge for 5 minutes at 6,000xg.
    7. Remove 950 μl of supernatant and resuspend the bacteria with 50 μl of this supernatant.
    8. Spread the solution on a LB agar plate containing the relevant antibiotic.
    9. Incubate in incubator at 37°C overnight.
    10. Check for colonies the next day.

    2. General bacterial concentration testing

    Materials:

    • Nano spectrophotometer
    • Test solution

    Protocol:

    1. Turn on the Nano spectrophotometer and select the measurement mode for bacterial suspension.
    2. Calibrate the spectrophotometer with standard samples to ensure accuracy.
    3. Add the bacterial suspension to a cuvette and place it in the spectrophotometer to measure the absorbance. Ensure cleanliness and optical path alignment.
    4. Record the concentration of the bacterial suspension.

    3. Bacterial plating

    3.1 Dilution coated plate method

    Materials:

    • Bacteria solution cultured for a period of time
    • Solid medium
    • Spreader

    Protocol:

    1. Add some bacteria solution onto the surface of the medium.
    2. Spread from one side of the plate, draw a few circles, and then overlap the streaks. Use a sterilized spreader and ensure it’s not too hot.
    3. Place the plates in the incubator with clean surfaces. Temperature varies by bacteria type.

    3.2 Plate streaking

    Materials:

    • Bacteria solution cultured for a period of time
    • Solid medium
    • Inoculating loop

    Protocol:

    1. Dip inoculating loop into some bacterial liquid.
    2. Streak on one side of the plate, do a quarter turn, and then overlap the streaks.
    3. Place the plates in the incubator with clean surfaces. Temperature varies by bacteria type.

    4. Phenotype inducing

    4.1 Induced by IPTG

    1. Culture the bacteria solution for 12 hours, ensuring OD600 is no less than 0.8.
    2. Add 16 μl of 50 mg/ml IPTG.
    3. After induction, culture at 37°C.
    4. Sometimes, a non-induced control group is used.

    4.2 Microscopy

    1. Observe smears using fluorescence and optical microscopes.

    Sudan Black Staining

    1. Prepare 5 g/L Sudan Black staining solution.
    2. Spread culture in thin film over slide.
    3. Dry in air.
    4. Pass slide through flame to fix.
    5. Flood slide with stain; rinse and dry.
    6. Place drop of oil on slide; examine with 100x objective.

    Triparental mating

    6.1 Preparation: Bacterial Strain Activation and Liquid Culture

    1. Activate the recipient strain S. fredii CCBAU45436 on TY+NA solid plates at 28°C for about 4-6 days until single colonies are visible. Inoculate the recipient strain to prepare the seed culture. (5-7 days before the experiment)
    2. Activate the donor strain on LB+Gen solid plates; activate the helper strain on LB+Km at 37°C; inoculate the recipient strain in TY+NA tubes and culture at 28°C with shaking at 180 rpm. (1-2 days before the experiment)
    3. Inoculate the donor strain in LB+Gen tubes and culture at 37°C with shaking at 200 rpm; inoculate the helper strain in LB+Km tubes and culture at 37°C with shaking at 200 rpm. (3-4 hours before the experiment)

    6.2 First Experiment

    1. Collect 1 mL of late-log phase recipient bacteria (rhizobia in TY medium, OD600 = 2~4), 0.5 mL each of mid-log phase donor and helper bacteria (E. coli in LB medium, OD600 = 0.5-1.0) in a 1.5 mL centrifuge tube, and centrifuge at 12,000 rpm or 13,000 x g at room temperature for 1 minute.
    2. Resuspend and mix all bacteria with 1 mL of sterile TY liquid medium or sterile saline (vortex to mix well), and centrifuge at 12,000 rpm at room temperature for 1 minute. Discard the supernatant to remove residual antibiotics from the medium.
    3. Repeat last step to wash the bacteria once more.
    4. Resuspend and combine all bacteria obtained from the centrifugation with 1 mL of saline (or TY liquid medium), and centrifuge at 12,000 rpm at room temperature for 1 minute.
    5. Retain about 30-50 μL of the supernatant, mix and vortex the bacterial pellet well, spot it onto the center of a TY agar plate without antibiotics; after the bacterial spot dries and stops spreading, invert and incubate at 28°C for 20-40 hours.

    6.3 Second Experiment

    1. Observe and record the growth of bacteria on TY+NA+Gen plates (complete photography and counting after subsequent sterile operations, select plates with a bacterial count between 30-300 for counting).
    2. Use sterilized white or yellow pipette tips to pick a small amount of single-crossover bacterial cells from the TY+NA+Gen plates (select from plates with lower colony density) and inoculate into TY liquid medium (without antibiotics or with NA only), culture at 28°C with shaking at 180 rpm for 24-30 hours.

    6.4 Third Experiment

    1. Observe and record the growth of double-crossover colonies on TY+sucrose+NA plates (photograph and count after completing subsequent sterile operations, select plates with a colony count between 30-300 for enumeration).
    2. Use sterilized toothpicks (white/yellow pipette tips) to randomly select 3 large colonies (wild type) and 3 small colonies (mutants), and transfer them to clean TY+NA+5% sucrose solid medium plates for incubation at 28°C for 2-3 days.

    Touchdown PCR

    Materials:

    • PCR tubes (200 μl)
    • 2 x Taq Mix
    • MucR1-WL (10 μM)
    • Template
    • ddH2O
    • Thermocycler

    Protocol:

    1. Add in a PCR tube:
      Substance Volume
      2 x Taq Mix 12 μl
      MucR1-WL (10 μM) 1 μl
      MucR1-WL (10 μM) 1 μl
      ddH2O 11 μl
    2. Use a white pipette tip to take a small amount of bacterial cells (0.1 μL) as a DNA template.
    3. Except for the template, mix in advance in an n-fold quantity and then aliquot.
    4. Place the tube in the thermocycler and start corresponding program.

    Agarose Gel Electrophoresis Analysis

    1. Gel Preparation (1.5%)
    2. Electrophoresis: 3-5 μl of sample, 5 μl of marker, constant voltage of 150V for 30-60 minutes.

    Plant Experiment

    1. Prepare Low-Nitrogen Nutrient Solution

    Prepare the low-nitrogen nutrient solution according to the following recipe for every 100 seedlings (500 mL needed).

    Substance Volume
    Ca(NO3)2·4H2O 0.6 g/L
    KCl 1.5 g/L
    MgSO4·7H2O 2.46 g/L
    K2HPO4·3H2O 3.56 g/L
    Microelement (1x) 20 ml/L
    CaSO4·2H2O 9.2 g/L
    Ferric citrate·5H2O 2.05 g/L

    2. Container-related Preparation

    1. Thread the cup with a gauze strip.
    2. Divide the water into portions and sterilize at high temperature for 30 minutes.
    3. Mix with vermiculite (1 large bag of vermiculite is needed for every 100 seedlings) and sterilize.

    3. Seed-related Preparation and Inoculate the Seedlings

    1. Select seeds (For planting x number of seedlings, it is best to select 3x number of seeds).
    2. Disinfect the seeds (with chlorine gas disinfection method).
    3. Prepare 0.8% water agar, sterilize, pour plates, and germinate seeds.
    4. Plant the seedlings. The device is shown in the figure below.
    5. Begin shaking the culture 24 hours before inoculation, and the OD value should be around 2. Collect the bacteria. Inoculate the bean seedlings.
    6. Regularly observe the growth, supplement water and nutrients as needed, adjust lighting, and harvest.

    Others

    1. Fluorospectrophotometer

    Materials:

    • F93 Fluorospectrophotometer (Lengguang Tech)
    • Test samples
    • Reference solution

    Protocol:

    1. Preheat the main unit for 30 minutes.
    2. Adjust to the required wavelength.
    3. Select the fluorescence value measurement function.
    4. Set sensitivity and zero adjustment.
    5. Open the sample chamber cover, remove cuvette one, place cuvette two into the cuvette holder, and close the sample chamber cover.
    6. Read and record the measurement.
    7. Proceed to measure other samples, or turn off the main power switch of the instrument.

    2. Mass Spectrum

    Materials:

    • Liquid nitrogen
    • Test samples
    • 50% methanol
    • Ultrasonic cell disruptor
    • Centrifuge
    • Filtration apparatus

    Protocol:

    1. Grind into a powder using liquid nitrogen.
    2. Weigh out an equal amount of the sample, 100mg, add 1ml of 50% methanol, and sonicate for 30 minutes.
    3. Centrifuge at 14,000 rpm for 10 minutes.
    4. Take the supernatant and filter it to obtain the filtrate, then perform a concentration gradient dilution with 50% methanol.
    5. Carry out direct mass spectrometry analysis. (Completed by the Mass Spectrometry Platform of the College of Biological Sciences, China Agricultural University.)

    3. Transmission Electron Microscope

    Materials:

    • Liquid nitrogen
    • Test samples
    • 50% methanol
    • Ultrasonic cell disruptor
    • Centrifuge
    • Filtration apparatus

    Protocol:

    1. S. fredii are cultured in TY broth without antibiotics until they reach the stationary phase.
    2. Centrifuge at 8,000xg for 5 minutes to collect 1 ml of bacterial cells.
    3. Discard the supernatant.
    4. Resuspend the cells in 2.5% glutaraldehyde for fixation.
    5. Proceed with subsequent transmission electron microscopy operations. (Completed by the Mass Spectrometry Platform of the College of Biological Sciences, China Agricultural University.)

    4. Thin-Layer Chromatography (TLC)

    Materials:

    • 0.1 M PBS
    • Methanol
    • Chloroform
    • 0.9% Sodium chloride solution
    • Nitrogen
    • n-Hexane
    • Methyl tert-butyl ether
    • Acetic acid
    • 8% Phosphoric acid
    • 10% CuSO4 · 5H20

    Protocol:

    1. Collect bacteria
      1. Collect 10 mL of bacterial solution into a pre-weighed centrifuge tube.
      2. Centrifuge at 8000 rpm for 1 minute to collect the bacterial cells.
      3. Wash twice with 0.1 M PBS.
      4. Freeze in liquid nitrogen and calculate the dry weight of the bacterial cells after vacuum freeze-drying.
    2. Extract oil
      1. Add 1ml of methanol to 25 mg of bacterial cells, and react for 5 minutes.
      2. Add 3ml of chloroform and 0.5 ml of 0.9% sodium chloride solution, shake and mix well.
      3. Centrifuge at 3000 rpm for 6 minutes.
      4. Transfer the lower organic phase to a pre-weighed glass test tube.
      5. Dry the organic phase with nitrogen, and weigh to calculate the amount of extracted oil.
    3. Chromatography
      1. Redissolve the grease with chloroform in the ratio of 1:20.
      2. Take 10 μl of spot sample to the Silica gel 60 F254 plate.
      3. After the spot sample is completed, prepare the developing agent: n-hexane: methyl tert-butyl ether: acetic acid=80:20:2 (v/v/v, currently prepared and used).
      4. Spread for 20 minutes, take out the plate 15 cm from the upper edge, and blow dry.
      5. Show the color in chromogenic solution (8% phosphoric acid and 10% CuSO4·5H20) for 30 seconds, take out and blow dry.
      6. Bake at 180 °C for 3 minutes.
      7. Record with the gel imager.