Experimental Objectives

By using bioinformatics methods and reference genome databases such as SoyBase and NCBI, design specific PCR primers for target genes and promoters, which will be used in subsequent molecular biology experiments including gene cloning and expression analysis.

Main Databases and Software

Databases

NCBI (National Center for Biotechnology Information): Used for sequence alignment verification and retrieval of published gene sequences.
SoyBase: A soybean genetic database.

Primer Design Software

Primer-BLAST: An online primer design tool that can complete both specific primer design and genome-wide specificity verification in one step.
OligoCalc: Used for online analysis of the physicochemical properties of primers.
Snapgene: Used for visual pairing of primers and target sequences.

Experimental Procedures

Obtain the sequences of target genes and promoters from the SoyBase database.
Target genes: Primers for GmGCH1, GmDHFR, GmADCS, and GmHPPK.
Promoters: Primers for GmLEA5, GmOleosin1, GmOleosin2, and GmLEA7.
Use the Primer-BLAST online primer design tool in the NCBI database to design primers.

Use the BLAST tool in the SoyBase database to verify the specificity of the primers.
Copy the screened primer sequences into OligoCalc, and reconfirm whether their Tm values, GC contents, and other parameters meet the requirements.

Reagents and Instruments Required for RNA Extraction

Reagents: Chloroform, isopropanol solution, RNase-free water, 75% ethanol
Instruments: Fume hood, vortex mixer, micropipette, refrigerated microcentrifuge

Preparations

Pre-cool the centrifuge.
Pre-cool the reagents (isopropanol, ethanol).

Experimental Procedures

1．Grinding: Put approximately 2 cm³ of young leaves and 2 steel balls (sterilized by ethanol burning) into a 2 mL RNase-free centrifuge tube. Grind the mixture using a grinder at 60 Hz for 120 seconds.
2．Add 1 mL of TRIzol reagent, shake well, and let it stand at room temperature for 5 minutes.
3．Add 0.2 mL of chloroform, mix thoroughly, and let it stand at room temperature for 3 minutes.
4．Centrifuge the mixture at 12,000 rpm and 4°C for 15 minutes.
5．Transfer 500 μL of the supernatant to a 1.5 mL RNase-free centrifuge tube, add 500 μL of isopentanol, shake well, and incubate in a 4°C refrigerator for 10 minutes.
6．Centrifuge the mixture at 12,000 rpm and 4°C for 10 minutes.
7．Aspirate and discard the supernatant, retain the precipitate, add 1 mL of 75% ethanol, and shake for washing.
8．Centrifuge the mixture at 7,500 rpm and 4°C for 5 minutes.
9．Aspirate and discard the supernatant, and air-dry for 10 minutes to completely remove ethanol.
10．Add 50 μL of RNase-free water and mix thoroughly to dissolve the precipitate.
11．Use a Nanodrop spectrophotometer to determine whether the OD260/280 ratio and OD260/230 ratio are within the reasonable ranges.
12．Store the extracted RNA in a -20°C refrigerator for later use.

Experimental Procedure

1．Design reverse transcription primers in accordance with the basic principles of primer design.
2．Prepare a system similar to the PCR system for amplification:

3．Mix the above system, transfer it to an EP tube, and place the tube in a PCR instrument for amplification. The PCR program is as follows:

4．Determine concentration and OD value:
a.Determine the concentration to confirm whether reverse transcription is successful.
b.Judge whether the OD260/280 ratio is within the reasonable range.

In this experiment, if the measured ratio is higher than 1.9, it may indicate that RNA has not been completely converted; if the measured ratio is lower than 1.8, the sample may contain impurities such as phenol or protein.
5．Store the final sample in a -20℃ refrigerator.

Reagents and Instruments

Reagents: 2×Phanta™ Master Mix, ddH₂O (double-distilled water), template DNA, primers

Instrument: PCR instrument

PCR Reaction System

PCR Amplification Conditions

Reagents and Instruments

Reagents:Agarose, 1× TBE buffer, nucleic acid dye (GelRed)

Instruments: Microwave oven, Erlenmeyer flask, gel tray, sample comb

Experimental Procedures

1．Weigh agarose:Weigh an appropriate amount of agarose according to the required concentration. For example, to prepare a 1% agarose gel, weigh 1 gram of agarose and place it in a 500-milliliter Erlenmeyer flask.
2．Add buffer: Add 100 milliliters of 1× TBE buffer to the Erlenmeyer flask, ensuring that the agarose is completely submerged in the liquid.
3．Heat to melt: Place the Erlenmeyer flask in a microwave oven and heat it on high power for 30 seconds. Take it out and shake gently to ensure that the agarose particles are fully dissolved. Repeat the heating and shaking process until the agarose is completely dissolved; usually, it needs to be heated to boiling.
4．Add dye: After heating until the agarose is completely dissolved, let it stand at room temperature to cool slightly. When the temperature drops to approximately 60℃, add 10 μL of the nucleic acid dye GelRed and shake thoroughly.
5．Pour the gel: Carefully pour the melted agarose solution into a pre-prepared gel tray, taking care to avoid generating air bubbles. When pouring the gel, the temperature should not be too low, otherwise the gel will solidify unevenly.
6．Insert the sample comb: Before the agarose gel is completely solidified, insert the sample comb, ensuring that there is an appropriate gap between the bottom of the comb and the bottom of the gel tray. 7．Cool and solidify: After the agarose gel is completely solidified, gently pull out the sample comb to form sample wells. Then add an appropriate amount of buffer to the tray, ensuring that the liquid level covers the surface of the gel.

Reagents and Instruments

Reagent: HiPure DNA Mini Column Kit (Brand: Magen Biotechnology Co., Ltd., Model: D2111)

Experimental Procedures

1．Prepare an agarose gel of appropriate concentration and perform electrophoresis to separate DNA fragments. After the DNA fragments are separated, place the gel under an ultraviolet lamp, quickly cut off the gel containing the target DNA fragment, and remove excess gel as much as possible.

2．Weigh the gel block and transfer it to a 2.0 mL centrifuge tube. Calculate based on the standard that 100 mg of gel block is equivalent to a volume of 100 μL, add 1.5 times the volume of Buffer GDP, and incubate in a 55℃ water bath for 15 minutes to completely dissolve the gel block. During the water bath, invert and mix 3 times to accelerate gel dissolution.

3．Perform a short centrifugation to collect the liquid droplets on the tube wall. Place the HiPure DNA Mini Column onto a collection tube. Transfer ≤700 μL of the gel-dissolved solution into the column, and centrifuge at 12,000×g for 30–60 seconds.

4．(Optional: If the gel-dissolved solution exceeds 700 μL) Discard the filtrate, place the column back onto the collection tube, transfer the remaining gel-dissolved solution into the column, and centrifuge at 12,000×g for 30–60 seconds.

5．Discard the filtrate, place the column back onto the collection tube, add 150 μL of Buffer GDP into the column, let it stand for 1 minute, and centrifuge at 12,000×g for 30–60 seconds.

6．Discard the filtrate, place the column back onto the collection tube, add 600 μL of Buffer DW2 (diluted with absolute ethanol) into the column, and centrifuge at 12,000×g for 30–60 seconds. Note: Before using Buffer DW2, dilute it with absolute ethanol according to the instructions on the bottle label.

7．Discard the filtrate, place the column back onto the collection tube, add 300 μL of Buffer DW2 (diluted with absolute ethanol) into the column, and centrifuge at 12,000×g for 2 minutes. When taking out the column, do not let the bottom of the column come into contact with the liquid; if it does, discard the liquid and centrifuge for another 1 minute. Open the lid of the column and air-dry for 5–10 minutes to completely remove ethanol.

8．Place the column onto a 1.5 mL centrifuge tube, add 15–30 μL of Elution Buffer to the center of the column membrane, let it stand for 2 minutes, and centrifuge at 12,000×g for 1 minute. Discard the column and store the DNA at -20℃.

Reagents and Instruments for Plasmid Extraction

Reagent: HiPure Plasmid Micro Kit (Brand: Magen Biotechnology Co., Ltd., Model: P1001)

Experimental Procedures

1．Isolate a single colony from a freshly streaked selective plate and inoculate it into 1–5 mL of LB medium containing the appropriate selective antibiotic. Incubate at 37°C for 12–16 hours with vigorous shaking. Centrifuge at 13,000 rpm for 1 minute at room temperature. Pour off or aspirate and discard the medium. It is strongly recommended to use endA-negative strains of E. coli for routine plasmid isolation. Examples of such strains include DH5α® and JM109®. The HiPure Mini protocol in this manual can be used for the preparation of low-copy-number plasmid DNA or cosmids. Use 1–10 mL of overnight cultured E. coli grown in LB medium. When a 10 mL culture volume is used, it is recommended to double the volumes of Buffer P1, P2, and P3.

2．Resuspend the pelleted bacterial cells in 250 μL of Buffer P1 and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should remain after this step. For resuspension of the pellet: Bacteria should be completely resuspended by vortexing. Mix by repeated pipetting until no cell clumps are left.

3．Add 250 μL of Buffer P2 and mix thoroughly by inverting the tube 8–10 times; gently invert to mix the liquid in the tube. Do not use a vortex mixer, as this will cause shearing of genomic DNA and contamination of the plasmid. Continue inverting the tube until the solution becomes viscous and slightly translucent. Do not allow the lysis reaction to proceed for more than 5 minutes.

4．Add 350 μL of Buffer P3. Mix immediately and thoroughly by inverting the tube 8–10 times. To avoid local precipitation, the solution should be mixed thoroughly immediately after adding Buffer P3. For large-volume cultures (e.g., ≥5 mL), inversion may need to be done 10 times. The solution should become turbid.

5．Centrifuge at 13,000 rpm for 10 minutes in a table-top microcentrifuge.

6．Insert a HiPure DNA Mini Column II into a 2.0 mL Collection Tube (provided). Pipette 800 μL of the supernatant from Step 5 onto the column. Centrifuge for 30–60 seconds. Discard the flow-through.

7．Recommended: Wash the column by adding 0.5 mL of Buffer PW1 and centrifuge for 30–60 seconds. Discard the flow-through. This step is necessary when using endA+ strains (e.g., JM series, HB101 and its derivatives) or any wild-type strains that have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5α do not require this additional washing step.

8．Wash the column by adding 0.75 mL of Buffer PW2 and centrifuge for 30–60 seconds.

9．Discard the flow-through and centrifuge at full speed for an additional 1 minute to remove residual wash buffer. Residual wash buffer will not be completely removed unless the waste flow is discarded before performing this additional centrifugation step. Residual ethanol in Buffer PW2 may inhibit subsequent enzymatic reactions.

10．Place the column in a clean 1.5 mL microcentrifuge tube. To elute the DNA, add 50–100 μL of Elution Buffer or water to the center of each column, let stand for 1 minute, and centrifuge for 1 minute. When the plasmid DNA or cosmids are larger than 10 kb, preheat the Elution Buffer (or water) to 70°C before use to elute the DNA from the HiPure membrane.

Experimental Reagents

Restriction enzymes: HindIII and XbaI (Brand: Yugong Biotechnology Co., Ltd.)
Corresponding buffer: 10×CutOne Color Buffer
PTF101 empty vector

Experimental Procedures

① Prepare the reaction system on ice according to the recommended sample addition order as follows:

② Mix by gently pipetting up and down or flicking the tube wall, then perform a brief centrifugation to collect the liquid droplets adhering to the tube wall.
③ Incubate at 37℃ for 15 minutes.
④ Incubate at 80℃ for 20 minutes to inactivate the enzyme.

Instruments for Electrophoresis Detection

Electrophoresis apparatus, developer Settings for electrophoresis apparatus: Voltage = 220 V, Time = 10 min

Reagents and Instruments for Gel Extraction

Reagent: HiPure DNA Mini Column Kit (Brand: Magen Biotechnology Co., Ltd., Model: D211)

Experimental Procedures

1．Prepare an agarose gel of appropriate concentration and perform electrophoresis to separate DNA fragments. After the DNA fragments are separated, place the gel under an ultraviolet lamp, quickly cut off the gel containing the target DNA fragment, and remove excess gel as much as possible.

2．Weigh the gel block and transfer it to a 2.0 mL centrifuge tube. Calculate based on the standard that 100 mg of gel block is equivalent to a volume of 100 μL, add 1.5 times the volume of Buffer GDP, and incubate in a 55℃ water bath for 15 minutes to completely dissolve the gel block. During the water bath, invert and mix 3 times to accelerate gel dissolution.

3．Perform a short centrifugation to collect the liquid droplets on the tube wall. Place the HiPure DNA Mini Column onto a collection tube. Transfer ≤700 μL of the gel-dissolved solution into the column, and centrifuge at 12,000×g for 30–60 seconds.

4．(Optional: If the gel-dissolved solution exceeds 700 μL) Discard the filtrate, place the column back onto the collection tube, transfer the remaining gel-dissolved solution into the column, and centrifuge at 12,000×g for 30–60 seconds.

5．Discard the filtrate, place the column back onto the collection tube, add 150 μL of Buffer GDP into the column, let it stand for 1 minute, and centrifuge at 12,000×g for 30–60 seconds.

6．Discard the filtrate, place the column back onto the collection tube, add 600 μL of Buffer DW2 (diluted with absolute ethanol) into the column, and centrifuge at 12,000×g for 30–60 seconds. Note: Before using Buffer DW2, dilute it with absolute ethanol according to the instructions on the bottle label.

7．Discard the filtrate, place the column back onto the collection tube, add 300 μL of Buffer DW2 (diluted with absolute ethanol) into the column, and centrifuge at 12,000×g for 2 minutes. When taking out the column, do not let the bottom of the column come into contact with the liquid; if it does, discard the liquid and centrifuge for another 1 minute. Open the lid of the column and air-dry for 5–10 minutes to completely remove ethanol.

8．Place the column onto a 1.5 mL centrifuge tube, add 15–30 μL of Elution Buffer to the center of the column membrane, let it stand for 2 minutes, and centrifuge at 12,000×g for 1 minute. Discard the column and store the DNA at -20℃.

Experimental Reagents

ClonExpress Ultra One Step Cloning Kit V2 from Thermo Fisher Scientific Inc.

Amount of Fragments

Optimal amount of cloning vector = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol)Optimal amount of insert fragment = [0.04 × number of base pairs of insert fragment] ng (0.06 pmol)

Experimental System

Gently pipette up and down to mix (do not vortex), then centrifuge briefly to collect the reaction solution at the bottom of the tube.Subsequently, incubate at 50°C for 5 minutes; cool down to 4°C or place on ice immediately for cooling.

Materials

Trelief 5α Chemically Competent Cells (from Tsc-col Qingke Biotechnology Co., Ltd.) ,Luria-Bertani (LB) Liquid Medium without Antibiotic Resistance

Experimental Procedure

1．Thaw the competent cells on ice.
2．Add 1 μL of recombinant plasmid to 20 μL of competent cells and mix gently.
3．Place on ice for 30 minutes, then incubate in a 42°C water bath for 45 seconds, and then take it out and place it on ice for 2 minutes.
4．Add 500 μL of LB medium without antibiotic resistance, place it in a 37°C shaker for 1 hour, and then perform plate spreading for screening.

Experimental Materials and Reagents

Instrumentation and Equipment

PCR Thermocycler, Electrophoresis Power Supply and Gel Tank, Agarose Gel Imaging System, Vortex Mixer, Microcentrifuge
Pipettes and Tips (10μL, 200μL, 1000μL), PCR Tubes (0.2mL)

Reagents

2×Taq Master Mix (PCR Premix): Contains Taq DNA Polymerase, dNTPs, Mg²⁺, and reaction buffer. Using the premix simplifies operations and reduces contamination.
Sterile Ultra-Pure Water
Primers: Target primers designed for the experiment, diluted to working concentration with sterile ultra-pure water.
Positive Control: Plasmid DNA or colonies known to contain the target fragment.
Negative Control: PCR system without template DNA (replaced with water).
LB Agar Plates: Agar plates with single colonies grown after transformation.
Reagents for Agarose Gel Electrophoresis: Agarose, Indicator, DNA Molecular Weight Standard (DNA Marker).

Experimental Procedures

Step 1: Pre-Experiment Preparation

1．Calculate and prepare the PCR Master Mix according to the number of required reactions. It is recommended to prepare 1-2 extra reactions to compensate for pipetting losses.

2．Vortex to mix the Master Mix thoroughly, centrifuge briefly, and aliquot 20μL into each PCR tube.

Step 2: Addition of Template (Colony)

1．Gently touch a single colony on the transformation plate with a sterile 10μL pipette tip. Stir the tip with the colony gently in the PCR tube containing the aliquoted Master Mix to fully suspend the bacterial cells in the liquid. Replace the pipette tip and repeat the above operation to pick the next colony.

2．Do not add any template to the negative control tube, or add an untransformed colony as the negative control.

3．Tighten the PCR tube caps and centrifuge briefly to concentrate the liquid at the bottom of the tubes.

Step 3: PCR Amplification

Place the PCR tubes into the PCR thermocycler and set the appropriate reaction program.

Step 4: Detection of PCR Products

1．Prepare an agarose gel of appropriate concentration.
2．Load the mixture into the gel wells. Meanwhile, load the DNA Molecular Weight Standard into an adjacent well.
3．Perform electrophoresis at an appropriate voltage until the bromophenol blue indicator migrates to 2/3 of the gel length.
4．Observe and record the results using the gel imaging system.

Step 5: Result Analysis

Positive Clones: A specific amplified band appears at the expected DNA fragment size, consistent with the size of the positive control band. Negative Clones:No bands appear, or only faint bands appear at the position of primer dimers (below approximately 100bp). Negative Control: No bands should appear. If bands appear, it indicates contamination in the reaction system, and the experimental results are unreliable.

Materials

Competent Cells: K599 (pSoup) Chemically Competent Cells (from AC1082, Weidi Biotechnology Co., Ltd.)
Medium: Leagene TY Agar Medium (TY Medium); Antibiotic-free TY Liquid Medium (for post-heat shock culture)

Experimental Steps

1．Thaw the Agrobacterium competent cells on ice.
2．Add 1 μg of plasmid to every 100 μL of competent cells, and mix quickly by pipetting.
3．Incubate on ice for 5 minutes → place in liquid nitrogen for 5 minutes → incubate in a 37°C water bath for 5 minutes → incubate on ice again for 5 minutes.
4．Add 700 μL of antibiotic-free TY liquid medium, culture in a 28°C shaker for 2 hours, then perform plate spreading for screening.

Reagents and Instruments Required for RNA Extraction

1．Seed Sterilization: Take out the screened and stored Huachun 6 seeds from the -20°C refrigerator, soak them in 75% ethanol for 1 minute, then wipe dry. Arrange the seeds in a single layer in a petri dish and place them in a desiccator in a fume hood. Put a 250 mL beaker in the desiccator, add 5 mL of concentrated hydrochloric acid to 100 mL of sodium hypochlorite, cover the desiccator for sterilization for 13.5 hours. Take out the seeds, blow them in a ultra-clean workbench, and store them in a 4°C refrigerator for later use.

2．Seed Soaking: Soak the soybeans in sterilized ddH₂O, and place them in a dark environment at 24°C for 10-16 hours.

3．Bacterial Solution Preparation:Take out the EHA105 bacterial solution transformed with the target plasmid, and add it to YEP liquid medium containing 100 mg/L Spe and 30 mg/L Rif. Set the constant temperature shaker at 28°C and 240 rpm for overnight culture. The next day, measure the absorbance value of the bacterial solution (OD600 = 0.8-1.0). Pour the bacterial solution into a sterile 50 mL centrifuge tube, centrifuge at 5000 rpm for 10 minutes. Pour off the supernatant, resuspend the bacterial cells with CCM liquid medium to make OD600 = 0.6-0.8, and store at 4°C for the next experiment.

4．Explant Preparation and Transformation:In the ultra-clean workbench, use tweezers and a scalpel to cut the hypocotyl of the soaked seeds, cut the seeds vertically along the cotyledons, and remove the young buds, epicotyls and seed coats on the cotyledons.

5． Inoculation and Co-Cultivation:Soak the explants in the prepared CCM resuspension, treat with ultrasound for 3 minutes, vacuum for 10 minutes, then shake at 120-150 rpm and 28°C for 40 minutes. After inoculation, pour out the bacterial solution, spread the explants on the CCM solid medium covered with filter paper using tweezers. Seal with medical tape, place in the tissue culture room, set the temperature at 22°C, culture in the dark for 48 hours, then culture in light for 3-5 days until the cotyledons turn completely green.

6．Shoot Induction:Use a scalpel to cut off the overgrown hypocotyls, insert the explants obliquely into the shoot induction medium, seal with medical tape, and place in the tissue culture room. Set the temperature at 22°C and the light time at 16/8 hours, and culture for 14 days. Transfer the explants to a new shoot induction medium in a sterile plate, discard the explants without clustered shoots, place 6-7 explants in each medium, seal with medical tape, and place in the tissue culture room for induction for 14 days.

7．Shoot Growth:Four weeks after shoot induction, discard the undifferentiated materials, cut off the cotyledons of the explants with clustered shoots, transfer them to the shoot growth medium, and make the incisions fit the medium. Place 6-7 explants in each petri dish, place in the tissue culture room, and replace the medium every 14 days.

8．Rooting:Use a scalpel to cut off the shoots with a length of more than 3 cm from the root of the tissue, soak them in auxin for 1 minute, then transfer them to the rooting medium, and place them in the tissue culture room to continue growing. When 5-6 roots grow and there are also lateral roots, gently take out the plants from the rooting medium, and carefully rinse off the medium on the roots with tap water. Transplant the seedlings into nutrient soil (vermiculite: substrate soil = 1:2) sterilized by high temperature and high pressure, place them in a heat-preserving and moisture-preserving seedling tray, and place the seedling tray in an incubator. Set the temperature at 26°C and the light time at 12/12 hours. Water with nutrient solution for cultivation until pod formation.

Reagents and Instruments for DNA Extraction

Reagents

CTAB (Hexadecyl Trimethyl Ammonium Bromide), Tris-HCl, EDTA, NaCl, β-Mercaptoethanol, Chloroform, Anhydrous Ethanol, ddH₂O

Instruments

Grinder (equipped with zirconia steel beads), 65°C Oven, Centrifuge, EP Tubes

Steps for DNA Extraction

1．Put 2 cm³ of young leaves into a tube, and add 3 zirconia steel beads.
2．Freeze quickly in liquid nitrogen.
3．Grind with the grinder at 120 Hz for 60 seconds by oscillation.
4．Add 500 μL of CTAB extraction solution.
5．Invert to mix, place in a 65°C oven for 30 minutes, and mix the solution at intervals.
6．Add 450 μL of chloroform, mix thoroughly, and centrifuge at 12,700 rpm for 15 minutes.
7．Pipette 350 μL of supernatant into a new EP tube, add 650 μL of anhydrous ethanol, invert to mix, and place at -80°C for more than 1 hour.
8．Centrifuge at 12,700 rpm at 4°C for 15 minutes, then discard the supernatant.
9．Add anhydrous ethanol, mix, centrifuge for 1 minute, and pour off the supernatant; centrifuge briefly, then aspirate the residual liquid with a pipette tip.
10．Let the white precipitate dry naturally, then add 40 μL of ddH₂O to dissolve it. The DNA extraction is completed.

Reagents and Instruments Required for Gene PCR Amplification

Reagents

2×Phanta™ Master Mix, ddH₂O, Template DNA, Primers

Instrument

PCR Thermocycler

PCR Reaction System

PCR Amplification Conditions

Reagents and Instruments Required

Reagents

5×TAE (Tris-Acetate-EDTA Buffer), Agarose, 5×Loading Buffer, Golden View (nucleic acid stain), DNA Marker

Instruments

Electrophoresis Power Supply, Developer (Gel Imaging System), Microwave Oven, Electronic Balance, Pipette, Gel Mold, Comb (10 μL volume)

Experimental Steps

1．Weigh 1 g of agarose with an electronic balance, add 100 mL of 5×TAE solution, and heat in a microwave oven until the agarose is fully dissolved.
2．When the solution cools to approximately 60°C, add 3.4 μL of Golden View and shake well.
3．Pour the solution into a gel mold, insert a 10 μL-volume comb, and cool at room temperature for 20 minutes (to solidify the gel).
4．Take the agarose gel out of the mold, place it in an electrophoresis tank, and submerge it in electrophoresis buffer.
5．Use a pipette to aspirate 10 μL of PCR amplification product (with loading buffer added in advance) respectively, and add them to the sample wells in order.
6．Use a pipette to aspirate 3 μL of DNA Marker and add it to a sample well.
7．Set the voltage to 180 V, and perform constant-voltage electrophoresis for 15 minutes.
8．After electrophoresis, place the agarose gel into the developer to observe whether the size of the target band is correct, so as to determine the positive status of the transformed seedlings.

Reagents and Instruments Required

Reagents

Disodium hydrogen phosphate (Na₂HPO₄), Sodium dihydrogen phosphate (NaH₂PO₄), Ascorbic acid, β-Mercaptoethanol, α-Amylase, Protease, Rat serum, Pure water

Instruments

50mL small glass bottles, Graduated cylinders, Beakers, Weighing paper, Weighing spoons, Precision balance, 2mL sterile centrifuge tubes, 3KD MWCO ultrafiltration tubes, Pipettes (with appropriate volume ranges), Sterile pipette tips, Small coffee bean grinder, Water bath/metal bath, Shaker, Refrigerated centrifuge

Experimental Steps

Step 1: Prepare Phosphate Buffer in Advance

Prepare a neutral buffer containing disodium hydrogen phosphate, sodium dihydrogen phosphate, ascorbic acid, and β-mercaptoethanol (100mL system):
1．Weigh 0.317g NaH₂PO₄ and 0.271g Na₂HPO₄, add approximately 80mL pure water, and adjust the pH to 7.
2．Add 0.5g ascorbic acid and 0.2mL β-mercaptoethanol, adjust the pH to 7 again, and bring the total volume to 100mL.

Step 2: Extract Folate Components

Preliminarily extract folate-containing mixtures from soybeans:
1．On the day of the experiment, grind soybeans into powder using a small coffee bean grinder (freshly ground).
2．Add 50mg (0.05g) soybean powder and 1mL phosphate buffer to each 2mL centrifuge tube, shake well, and immediately transfer to a 100°C boiling water bath/metal bath for 10 minutes (during heating, take out and invert to shake evenly every 5 minutes to prevent clumping).
3．After boiling, open the tube cap to release gas, then close the cap and place the tube on ice for 10 minutes.

Step 3: First Enzyme Treatment

Add 20µL α-Amylase (0.1mg/mL) to the sample tube, and incubate in a 28°C shaker for 30 minutes.

Step 4: Second Enzyme Treatment

1．Pre-cool the centrifuge and set the centrifugation temperature to 4°C.
2．Add 15µL protease to the sample tube, continue incubating in a 37°C shaker for 1 hour.
3．After incubation, boil the sample in a boiling water bath for 5 minutes, then transfer to the 4°C centrifuge and centrifuge at 13,000 rpm for 10 minutes.
4．Aspirate 400µL of the supernatant into a new 2mL sterile centrifuge tube.

Step 5: Depolymerase Treatment

Add rat serum at a volume ratio of 2% to the supernatant, and incubate at 37°C for 4 hours to remove the polyglutamate tail.

Step 6: Washing and Centrifugation

1．Boil the sample again for 10 minutes, then cool it on ice for 10 minutes.
2．Centrifuge at 13,000 rpm for 10 minutes at 4°C.

Step 7: Sample Filtration and Preparation

1．Transfer 400µL of the supernatant to a 3KD MWCO ultrafiltration tube, centrifuge at 13,000 rpm for 20 minutes at 4°C, and aspirate 240µL of the supernatant into a new tube.
2．Finally, take 100µL of the filtrate for analysis, and store the remaining part at -80°C

Preparation of LB Antibiotic-Free Liquid Medium (1L)

Components & Dosages

Preparation Steps

1．Weigh 10 g TRYPTONE, 5 g Yeast Extract, and 10 g NaCl into a heat-resistant container, then add approximately 800 mL ddH₂O to dissolve them.
2．Bring the volume to 1 L, mix well, dispense into autoclave-resistant containers or bottles, and leave the lids loose.
3．Autoclave at 121°C for 15–20 minutes, then take out and cool to room temperature for later use.

Preparation of LB Antibiotic-Free Solid Medium (1L)

Components & Dosages

Preparation Steps

1．Weigh 10 g TRYPTONE, 5 g Yeast Extract, 10 g NaCl, and 14 g Agar into a container, add approximately 800 mL ddH₂O, heat and stir until fully dissolved (avoid local gelatinization).
2．Bring the volume to 1 L, dispense into containers, and autoclave at 121°C for 15–20 minutes.
3．Keep the sterilized medium warm for later use; cool it to approximately 55°C when antibiotics need to be added subsequently.

Extraction of Kanamycin (50 mg/mL)

1．Take a 50 mL tube, add 1.5 g kanamycin sulfate powder (molecular weight: 582.58) to 30 mL sterile ddH₂O.
2．Transfer to a sterile operating bench, draw the liquid with a syringe, replace with a filter tip, and filter the liquid into 2 mL sterile centrifuge tubes or other containers.

Preparation of LB Antibiotic-Containing Liquid Medium (1L)

Add 1 mL of kanamycin (Kana) working solution to the cooled LB antibiotic-free liquid medium.

Preparation of LB Antibiotic-Containing Solid Medium (1L)

1．When the LB antibiotic-free solid medium cools to approximately 55°C, add 1 mL of kanamycin (Kana) working solution.
2．Mix well, immediately pour into petri dishes (about 10 mL per dish), and store in a sterile operating bench.