Material



The reaction mix required for Taq and LAmp enzyme



The reaction mix required for pfu enzyme

Procedure
1. Prepare the total reaction mix based on the enzyme being used and total volume of PCR needed.
2. Vortex and then centrifuge the reaction mix for about one minute.
3. Separate the total reaction mix into several equivalent one if needed. Then, vortex and centrifuge them for about one minute. Note: Components common to all reactions can be combined and aliquoted individually (e.g. for reactions using the same REs but different inserts, combine water + buffer+ ligase + REs, aliquot into each tube, then add the different inserts)
4. Then set the temperature for annealing and the time for extension in the corresponding PCR systems.

Material
Primers
Enzymes and their corresponding buffer mix
ddH₂O

Procedure
Oligo pool
1. Add primers of 2μL each into a PCR tube.
2. Vortex and centrifuge.

First round of PCR
1. For the first round of PCR, add reagents according to the amounts given in the manual book.





2. Vortex and centrifuge.
3. Set the temperature and time for PCR.

Second round of PCR
1. Use the product of the first round of PCR as the template to perform a second round of PCR
2. Add reagents into other PCR tubes for the second round of PCR.





Gel electrophoresis and gel extraction (Refer to the sections of Gel Electrophoresis and Gel extraction)
1. Chose the appropriate gel size with large wells for gel electrophoresis and gel extraction.
2. When PCR ends, add 5μL 10x Loading buffer, then vortex and centrifuge.
3. Load 5μL of DNA marker to the first well of each piece of gel. Then load the rest of the 50μL PCR product and loading buffer into the wells.
4. Set voltage to 140V and run for 20 minutes.
5. After Gel electrophoresis, perform gel extraction according to the gel extraction section.

Material
Transformed plates
LB medium
Primers
PCR master mix
ddH₂O

Procedure
1. Prepare a replica plate using the agar plate corresponding to colony resistance. Label the plate with a reference table to help align the colonies.
2. Prepare the reaction mix based on the following table:



3. Pick one colony and dissolve in the reaction mix.
4. Transfer 0.5 μL onto the labelled replica plate, grow the plate overnight in 37°C incubator.



*L = length of fragment, R = rate of polymerase
5. Final hold temperature at 4°C
6. Run agarose gel electrophoresis on the PCR samples (verification, 3 μL/well)
7. Send the remaining PCR product with its primers for sequencing

Material
Centrifuge
Dry bath incubator
CA2 spin column
Collection tube
Buffer BL
Buffer PN
Buffer PW
ddH₂O
1.5mL centrifuge tube

Procedure
1. Excise desired DNA band from agarose gel and place it into a 1.5 mL centrifuge tube
2. Measure the mass of the DNA band
3. Add X μL of buffer PN (magnitude of X=100 x mass of gel excised)
4. Place the centrifuge tube in 60°C 800rmp dry bath incubator to liquify the gel
5. Add 500μL of buffer BL into a CA2 spin column placed in a collection tube
6. Centrifuge for 1min at 12,000rpm
7. After gel is fully molten, cool to room temperature
8. Add the gel solution into the CA2 spin column
9. Place at room temperature for 2min
10. Centrifuge for 1min at 12,000rpm
11. Add the liquid left in the collection tube back into the spin column
12. Place at room temperature for 2min
13. Centrifuge for 1min at 12,000rpm
14. Add 600μL of buffer PW into the spin column
15. Centrifuge for 1min at 12,000rpm
16. Discard the liquid in the collection tube and put the spin column back into the collection tube
17. Repeat steps 15-16
18. Centrifuge the spin column and collection tube for 2min at 12,000rpm
19. Place the collection tube into a 1.5mL centrifuge tube
20. Open the lid of the spin column and place the centrifuge tube in 60°C dry bath incubator for 5min
21. Add 35μL $$ddH_2O$$ onto the membrane of the CA2 spin column
22. Close the lid of the spin column and place the centrifuge tube in 60°C dry bath incubator for 2min
23. Centrifuge for 2min at 12,000rpm
24. Add content of the collection tube back into the spin column and repeat step 23
25. Discard the collection tube, the liquid collected in the centrifuge tube is the DNA solution
26. Use ELISA reader to measure the concentration of the DNA solution
27. Store the DNA solution in -20°C fridge

Apparatus and materials
Apparatus
Casting/gel tray
casting stand
well combs
voltage source
gel box
microwave oven
pipette
pipette heads

Materials
TAE buffer
agarose
syBr

Procedure


Table 1.1 Composition for different gels

1. Based on your intention (verification or gel recycling), and number of samples, choose the appropriate well comb(s) and casting tray based on the flowchart below:



2. Place casting tray into casting stand, then slide in well combs into slots for the comb on the casting stand
3. Add the according amount of TAE buffer from table 1.1 into a conical flask using a measuring cylinder, be certain that it was placed within the “gel area”, not from open bench lab/living area outside the contaminated area
4. Using weighing paper, plastic spatula, and electronic balance, weight and add the according amount of agarose from table 1.1 into the conical flask, be sure no agarose is stuck on the walls of the flask, swirl the flask
5. Heat the conical flask using the microwave oven, set power to medium-high
6. When the solution in the flask boils (i.e. lots of visible bubbles), remove the flask from the oven using a baking glove, swirl the flask to mix the solution for ~10 seconds
7. Place the flask back into the oven, wait for the solution to boil again 2 times, remove the flask from the oven using the baking glove, under direct light, see if the solution is clear all the way through, with no visible, feather-like precipitate, if not, continue to heat the solution until no precipitate could be seen
8. Cool the solution by washing the exterior of the flask using cold water, cool until no more vapor could be seen and the flask do not feel too hot to be touched with rubber gloves
9. Using a pipette, add according amount of gel dye from table 1.1 into the flask, swirl the flask to ensure the solution is evenly mixed
10. Pour the solution into the prepared casting tray
11. Wait around 20 minutes for the gel to solidify
12. Place the casting tray into the gel box, ensure surface of the solidified gel is totally submersed below TAE buffer
13. Load the appropriate DNA ladder into the first well, starting from the left
14. Load the samples into the rest of the wells (add DNA loading buffer to the samples if haven’t already)
15. Set parameters of voltage/power source to 20 min, 400 mA, 140V
16. Put back the lid, ensure the electrodes are matched, run the gel by pressing the start button on the voltage source
17. After 20 minutes, results could be visualized by shining UV/blue light to the gel
18. The gel, after taking a picture/have the fragments of correct length cut away, should be disposed into trash bins within the gel area, do not take the gel outside the gel area

Procedure



1. Use the algorithm to calculate the volume of vector and inserts that should be added:



C1, V1, and L1 represents concentration, volume, and length of the vector respectively. If all the reaction mixtures are fragments, the longest one will be the vector of this algorithm. The volume of the vector will usually be initiated as 1ul, though the volume depends on the concentration of the vector(C1V1 >= 100). Meanwhile, C2, V2, and L2 connote the concentration, volume, and length of the inserted fragment. The variable n can be switched considering the ratio between the length of the vector and the fragments.
2. After calculation, assemble all the components together inside a PCR tube.
3. Mix the component through vortex.
4. Centrifuge the tube in order to concentrate the liquid at the bottom.
5. Place the PCR tube into a thermocycler and start the following program:




DNA Concentration Calculator
You can use this DNA Concentration Calculator to calculate the correct volume of each component to add to the reaction onto the replica

Procedure
1. Assemble all reaction components in appropriately labelled PCR tubes. Components common to all reactions can be combined and aliquoted individually (e.g. for reactions using the same REs but different inserts, combine water + buffer+ ligase + REs, aliquot into each tube, then add the different inserts)
2. Mix the liquid via vortex, collect all the liquid to the bottom of the tubes by centrifuging for a few seconds
3. Place the PCR tubes into a thermocycler and run the below program

DNA Concentration Calculator
You can use this DNA Concentration Calculator to calculate the correct volume of each component to add to the reaction onto the replica

Materials
Foam ice box
Metal path pot
Clean bench
Constant temperature shaker
Thermostatic incubator
E. coli competent cells
Plasmid
LB liquid medium
L-shaped cell spreaders

Procedure
1. Thawing: Remove the E. coli competent cells from the -80 ° C freezer and thaw on ice (approximately 1 minute).
2. Distribution: E. coli competent cells 100μL/tube. Label the newly taken clean 1.5 mL centrifuge tube, take out 40 mL of thawed receptive cells, and divide them into clean centrifuge tubes to obtain 2 portions of converted receptive cells.
3. Ice bath: Mix 1μL plasmid (c>100 ng/μL) or all connecting systems with 30-50μL competent cells and incubate in ice for 20-30 minutes.
4. Heat Shock: Adjust the metal bath to 42 ° C and heat shock at 42 ° C for 90 seconds after the ice bath is over.
5. Ice bath 2: Put it on ice for 5min
6.Recover: Add 300μL of non anti LB culture medium to each tube of competent cells in the ultra clean workbench. Fix the receptive cells in each tube in a centrifuge tube floating plate and place them on a shaking table at 37 ° C to revive the bacteria for 30 minutes-1hour
7. Coating: In the ultra clean workbench, take 150-200μL of bacterial solution and add it onto the agar plate. Use a coating rod to evenly apply the bacterial solution onto the LB agar plate containing antibiotics. After the bacterial solution on the LB plate is dried, place the plate upside down in a 37 ° C incubator and incubate for 16-20 hours, then observe the clones.

Apparatus and materials
Apparatus
Centrifuge
Benchtop
Laboratory thermo shaker
Ultrasonic cell disruptor
Ice maker

Materials
Glycerobacterium
Cultural dishes with antibiotics
corresponding antibiotic plates
Anbiotic
Inducer
20mM Tris-HCI pH8.0

Procedure
Preparation of microbes
1. Remove the centrifuge tube containing glycerolbacterium of your strain from the -80°C freezer
2. Thaw and extract 5μL of frozen glycerobacterium in clean bench.
3. Inoculate on the appropriate antibiotic plate and culture at 37°C overnight (~16h) until single clones appear.

Pre-culture of microbes
4. Dispense 4-5mL of LB liquid medium with the corresponding antibiotic into a 15mL culture tube
5. Pick a single clone to inoculate into the liquid medium in the culture tube.
6. After inoculation, place the culture tube into a thermal incubator set to an appropriate temperature, i.e., 37°C, on a shaker at 220rpm for overnight cultivation (~16-20 hours).

Seed-culture
7. Prepare a sterilized empty conical flask and add an appropriate amount of LB medium (or primary culture medium).
8. Take the overnight pre-cultured bacterial strain and inoculate it into the conical flask at a ratio of 1:100 volume. If the primary culture medium is not LB, centrifuge the pre-culture suspension at 7500rpm, discard the LB medium, resuspend at an equal volume of primary culture medium, and then proceed with inoculation.
9. After inoculation, culture at 37°C on a shaker at 220 rpm until the OD600 reaches between 0.6-0.8. A simple method to determine this is to hold the bottom of the conical flask by hand, tilt it at a 45° angle, and observe from the top down. If you can see the outline of your fingers through the solution but can hardly make out the details of the finger grooves, then the OD value is likely around 0.6-0.8.

Induction of Microbial Expression
10. During induction, add an appropriate amount of IPTG to the conical flask to achieve a final concentration of 0.3 mM. This means adding 15 μL of 1M IPTG to the 50 mL induced expression system. The formula for calculating the amount of IPTG to add is the following:



In which C1 refers to the initial concentration of IPTG; C2 refers to the target concentration (0.3 mM) of IPTG; V2 refers to the volume of the induced expression system, and V1 refers to the volume of IPTG required to achieve the target concentration.

Main-culture of microbes
11. Place the conical flask in a shaking incubator at a lower temperature for the primary culture. Typically, overnight cultivation for protein expression is done at a temperature of 10-20°C with shaking at 220 rpm for 16-20 hours. For high-temperature induction, you can try increasing the temperature to 30-37°C and continue shaking at 220 rpm for 1-4 hours.

Apparatus and materials
Apparatus
Centrifuge
Benchtop
Ultrasonic cell disruptor
Ice maker

Materials
Collected liquid culture of microbial cell
20mM Tris-HCI pH8.0
Loading buffer

Procedure
Sonication cell lysis
1. Take an appropriate amount of Tris-HCl or PBS solution as the lysate solution for resuspending the microbial cells. For every 50mL of bacterial cells, add 5mL-10mL of the lysis solution for resuspension. If the proteins are sensitive to salt, you can resuspend in a 20mM Tris-HCl pH 8.0 solution only. For proteins that are not sensitive to salt, you can add 300mM NaCl to the buffer.
2. Vortex mixes the bacteria with the lysate evenly, and optionally adds a suitable amount of protease inhibitor. Microorganisms that will be processed soon can be stored at -20°C.
3. Prepare a beaker filled with ice and add a 1:2 ratio of water to ice to create an ice-water mixture.
4. Draw 1ml of bacterial cells into a 2ml centrifuge tube.
5. Place the 2ml centrifuge tube in the beaker and secure it with ice to keep the liquid below the level of the ice-water mixture. The probe of the ultrasonic disruptor needs to be completely immersed in the lysis solution but should not touch the walls or bottom of the tube. Maintain a distance of 1cm between the probe and the bottom of the tube.
6. The disruption program is set at 60W power, 25kHz frequency, with the ultrasound on for 3 seconds and off for 6 seconds, in cycles repeated for 5 minutes. The completion of ultrasonication is indicated by a clear and foam-free liquid.

Sample Preparation
7. Subsequently, centrifuge at 12,000 rpm for 2 minutes.
8. Carefully transfer the supernatant into a new centrifuge tube and resuspend the pellet in a 20mM Tris-HCl pH 8.0 solution to the same volume as the supernatant.
9. Draw 16 μL of each of the supernatant sample ("supernatant") and the pellet sample ("precipitate") from the centrifuge tube to a PCR tube. In each PCR tube, add 4 μL of loading buffer.
10. Insert the PCR tubes into the PCR system and denature at 95°C.

Reagent Preparation

• Washing Buffer: 20 mM Tris HCl
• Elution Buffer: 200 mM Imidazole
• 20% Ethanol

Column Preparation
Warning: The liquid in the column must not dry out!!

1. Fill the column with 3 mL of Resin column material. The Resin column material can be reused; store in 20% ethanol at 4°C.
2. Open the stopper and let the ethanol in the column material flow out naturally.
3. Add 20 mM Tris-HCl to fill it up, let it flow out, and repeat 3 times to wash out residual ethanol.
4. Seal the stopper for later use.

Purification

1. Add the supernatant to the column material, collecting the outflow in a 50 mL centrifuge tube. The collected liquid is the flow-through (FT); observe if the FT becomes clear. If not, re-add to the column material until the flow-through is clear; repeat flow-through as much as possible.
2. Fill the column material with 20 mM Tris-HCl, open the stopper and collect the first 5 mL, mark the collected washing as W, the rest of washing is not needed. Repeat washing twice.
3. Fill the column material with 200 mM Imidazole, open the stopper and collect the first 3 mL, mark the collected elution as E, the rest of elution is not needed.

Post-Processing of Column Material

1. Wash with 200 mM Imidazole again, 2-3 times the column volume, to remove residual proteins.
2. Wash with 20 mM Tris-HCl, 4-5 times the column volume.
3. Wash with 20% ethanol, 2-3 times the column volume, then fill with 20% ethanol and store in the refrigerator at 4°C.

Warning: Ensure the ultrafiltration membrane has been cleaned with NaOH and pure water before use!

1. Add the elution to the upper layer of the ultrafiltration membrane.
2. Place the ultrafiltration membrane in a pre-cooled centrifuge at below 10°C and centrifuge at 6500 rpm for 10 minutes.
3. Transfer the filtrate from the lower layer of the ultrafiltration membrane to a new 50 mL centrifuge tube, mark it as filtrate, and transfer 1 mL into a clean 1.5 mL centrifuge tube.
4. Fill the upper layer of the ultrafiltration membrane with 20mM Tris-HCl to the mark.
5. Repeat steps b-d until the volume of the filtrate reaches 50 mL.
6. Transfer the upper layer liquid from the ultrafiltration membrane to a new 15 mL centrifuge tube.
7. Run the flow-through, wash, elution, and filtrate on a protein gel for verification.

1. Add the elution to the dialysis bag.
2. Soak the dialysis bag in 20mM Tris-HCL.
3. Stir with a glass rod every 10 minutes, renew 20mM Tris-HCL every 30 minutes.
4. After 3 hours of dialysis, collect the elution and run SDS-PAGE of flow-through, wash, elution.

1. Prepare the SUMO cleavage system on ice.
   • 10× SUMO Buffer (+Salt/-Salt): 300 µL
   • Sumo protease: 5 µL
2. Add the cleavage system to supernatant.
3. Incubate at 4°C overnight.
4. Validate the results by protein electrophoresis.

Apparatus and materials
Apparatus
Spacer Plate
Short plate
Casting frame
Gel Cassette Assembly
Casting Stand
Gel Cassette Sandwich
Buffer Dam
Electrode Assembly
Companion Assembly
Mini Tank and Lid

Materials
Beyotime SDS-PAGE Gel Quick Preparation Kit

• 30%Acr-Bis(29:1)
• 30%Acr-Bis(29:1)
• Upper gel buffer(4×)
• Gel polymerization catalyst 10%APS
• TEMED Substitute
• Instruction booklet

Loading buffer
Protein marker
SDS-PAGE running buffer (1×)
Commassie Blue Staining Solution

Procedure
Preparation of Glass Cassette
1. Prepare short plate and spacer plate, ensure the plates are clean and dry
2. Select a spacer plate of the desired thickness and place a short plate on top of it
3. Slide the spacer plate and short plate into the casting frame (with pressure cams open), ensure short plate is facing front of the casting frame and both plates are flush at the bottom
4. Secure the two plates by engaging pressure cams on the casting frame
5. Place the casting frame upright on to the casting stand by holding open the lever at top of the stand
6. Ensure backside of the casting frame is in contact with front of the casting stand along all its length, and the glass cassette’s bottom is pressed into the spacer plate
7. Fill the glass cassette with distilled water, leave the glass cassette in its stand for ~5 minutes
8. Check if the level of water in glass cassette dropped. If so, there is a leakage, and steps 1 to 7 shall be conducted again. If not, pour away all water in the cassette, dry the cassette using tissue if neccessary

Preparation of Separating Gel
9. Add necessary reagents from the tables below for separating gel to a 50ml centrifugal tube, mix the solution by gently turning the tube up-side-down and back again for a few times only add TEMED and APS after all other reagents are added and mixed
Protein Size and Corresponding Separating Gel Concentration:



Separating Gel Recipe:



10. Using a 1000μl pipette, inject separating 4ml of gel solution prepared into the glass cassette slowly, avoid forming an uneven boundary of the gel
11. Using a 1000μl pipette, inject 2ml distilled water or ethanol to seal off separating gel, while waiting for separating gel to solidify (usually take 25-30 minutes), continue to prepare stacking gel

Preparation of Stacking Gel
12. Add necessary reagents from the table below for separating gel to a 50ml centrifugal tube, mix the solution by gently turning the tube up-side-down and back again for a few times only add TEMED and APS after all other reagents are added and mixed
Stacking Gel Recipe:



13. After separating gel solidifies, pour away ethanol or distilled water, and then inject 1.5-2m stacking gel solution using 1000μl pipette, then place an appropriate comb into the cassette

Running the Gel
14. Remove the comb once the gel has solidified. Carefully remove the gel cassette sandwich from the casting frame
15. Place the first gel sandwich with the short plate facing inward onto the gel supports. The same goes with the second. As both cassettes tilt in 30 degrees away from the center of the frame, use one hand to squeeze both cassettes against the green gasket, and the other to clamp the clamping frame over the gels, locking them into place Note: There are two supports in each side of the clamping frame assembly, in which it requires 2 gels to create a functioning assembly, thus attempt to run an even number of gels at a time. If not, a buffer dam is required
16. Settle the electrode assembly into the mini-tank, fill the assembly with buffer to just under the edge of the outer gel plate
17. Load samples into each of the wells with a 10 µL pipette with 5 µL of sample per lane Note: sample loading should be conducted at a fairly slow speed to ensure the samples settle on the bottom of the well
18. Fill the tank with buffer to the indicated level. (550 ml for 2 gels)
19. Place the lid with power cables onto the mini tank with the electrode assembly inside. Ensure that the color of the wire on the lid matches the color mark on the tank accordingly
20. Insert the electrical leads into a power supply. 150 V and 50 minutes is the recommended setting for most SDS-PAGE gels

Gel Staining and Result Visualization
21. Gently remove the gel from the glass cassette and place it on a clear container; rinse the gel with tap water at least twice
22. Submerge the gel under SDS-Page stain and place it on a square magnetic stirrer to shake for 10 minutes
23. Remove the gel from the stirrer, rinse it with tap water. The results can thus be visualized, to obtain more precise readings, a gel imager could be used

Apparatus and materials
Apparatus
Centrifuge
Vortex mixer
Dry bath incubator
Column tube CP3
Collection tube
1.5 ml centrifuge tube
2 ml centrifuge tube

Materials
Buffer P1
Buffer P2
Buffer P5
Buffer PWT
Deionized distilled water
Inoculated Bacteria (minimum 4 ml)

Procedure
1. Collection of deposits of bacteria:Use a pipette to draw 2 ml of the inoculated bacterial fluid into the labeled 2 ml centrifuge tube accordingly. Centrifuge with parameter 12000 rpm for 1 minute. Remove the supernatant solution and repeat the steps with another 2 ml of the inoculated bacteria.
2. Mixture: Draw 150 µL of Buffer P1 into the 2 ml centrifuge tube and place the centrifuge tube on the vortex mixer to mix the solution until the precipitated bacteria is thoroughly suspended in the buffer. The mixed solution should appear as turbid red.
3. lkaline lysis:Draw 150 µL of Buffer P2 into the 2 ml centrifuge tube and mix the solution by gently inverting the tube for 6-8 times. The resulting liquid should appear clear purple. If traces of red can still be observed, repeat inverting until thorough purple is achieved.
4. Neutralization Renaturation:Within two minutes after the addition of Buffer P2, draw 350 µL of Buffer P5 into the centrifuge tube, and mix the solution by gently inverting the tube for 12-20 times. Flocculent precipitate should be observed in the clear yellow solution. If there are purple leftovers observed, then the previous step should be continued until thorough yellow is achieved.
5. Centrifuge with parameters 12000 rpm for 7 minutes.
6. Draw the supernatant from the centrifuge tube into the labeled spin column CP3 accordingly. Place the column tube CP3 into the according collection tube and centrifuge with parameters 12000 rpm for 30 seconds. Remove liquids from the collection tube.
7. Draw 300 µL of Buffer PWT into the column tube CP3, centrifuge with parameters 12000 rpm for 30 seconds. Remove the fluids from the collection tube.
8. Place the column tube CP3 into the collection tube and centrifuge for 2 minutes.
9. Place the column tube CP3 into the labeled 1.5 ml centrifuge tube accordingly, open the lids of the column tubes and place them in 60°C dry bath incubator for 3 minutes.
10. Add 50 µL of $$ddH_2O$$into the column tube CP3, close the lid and place it in 60°C dry bath incubator for 5 minutes.
11. Centrifuge the column tubes contained in 1.5 ml centrifuge tubes with parameters 12000 rpm for 1 minute.
12. Utilize the ELISA reader to measure the concentration of the extracted plasmid
13. Store the extracted plasmid in -20°C fridge

Apparatus and materials
Apparatus
Thermal cycler
rCutSmart
Restriction Enzyme
DNA
ddH₂O
Isopropanol
Centrifuge
Dry bath incubator
Spin column
Collection tube
Buffer BL
Buffer PN
Buffer PW

Procedure
Digestion
1. Prepare the reaction mix based on the following table:



2. Incubate the reaction at digestion temperature (usually 37 °C) for 1 hour.
3. Stop the digestion by heat inactivation (65 °C for 15 minutes)
4. The digested DNA is ready for use in research applications.

When using two restriction enzymes at once, first check the enzyme activities in each buffer, using the table on the Restriction Enzyme Buffer Reference. If they both have 100% activity in the same buffer, you can proceed with your double digestion protocol using that buffer. Alternatively, the optimal buffer can be determined from the chart of common double digestions. In some cases, sequential digestion is recommended due to buffer incompatibility (composition or temperature).

Purification
1. Excise desired DNA band from agarose gel and place it into a 1.5 mL centrifuge tube
2. Measure mass of the DNA band
3. Add X μL of buffer PN (magnitude of X=100 x mass of gel excised)
4. Place the centrifuge tube in 60°C 800rmp dry bath incubator to liquify the gel
5. Add 500μL of buffer BL into a CA2 spin column placed in a collection tube
6. Centrifuge for 1min at 12,000rpm
7. After gel is fully molten, cool to room temperature
8. Add the gel solution into the CA2 spin column
9. Place at room temperature for 2min
10. Centrifuge for 1min at 12,000rpm
11. Add the liquid left in the collection tube back into the spin column
12. Place at room temperature for 2min
13. Centrifuge for 1min at 12,000rpm
14. Add 600μL of buffer PW into the spin column
15. Centrifuge for 1min at 12,000rpm
16. Discard the liquid in the collection tube and put the spin column back into the collection tube
17. Repeat steps 15-16
18. Centrifuge the spin column and collection tube for 2min at 12,000rpm
19. Place the collection tube into a 1.5mL centrifuge tube
20. Open the lid of the spin column and place the centrifuge tube in 60°C dry bath incubator for 5min
21. Add 35μL ddH₂O onto the membrane of the CA2 spin column
22. Close the lid of the spin column and place the centrifuge tube in 60°C dry bath incubator for 2min
23. Centrifuge for 2min at 12,000rpm
24. Add content of the collection tube back into the spin column and repeat step 23
25. Discard the collection tube, the liquid collected in the centrifuge tube is the DNA solution
26. Use ELISA reader to measure the concentration of the DNA solution
27. Store the DNA solution in -20°C fridge

Ligation
1. Prepare the reaction mix based on the following table:



2. Incubate the reaction at digestion temperature (usually 37 °C) for 1 hour.
3. Stop the digestion by heat inactivation (65 °C for 15 minutes) or addition of 10 mM final concentration EDTA.
4. Recover the DNA using a purification kit: re-suspend and dilute the DNA to 1 µg/µL.
5. Prepare second digestion according to step 1. Continue through step 3.
6. The digested DNA is ready for use in research applications.

All operations with potential risks of contaminating the culture should be conducted in a safety cabinet.

1. Activation of Strain:
- Take 5 µl ofPichia pastoris SMD1168H glycerol stock stored at -80°C and streak on a YPD (+2% Glucose) plate. Incubate at 30°C for 2-4 days to activate the strain.
- Use: "47.5 ml YPD + 2.5 ml 40% Glucose"
2. Inoculation:
- Inoculate a fresh yeast colony into 5 ml YPD (+2% Glucose) liquid medium.
- Use: "47.5 ml YPD + 2.5 ml 40% Glucose"
3. Cultivation:
- Incubate at 28-30°C, 250 rpm overnight.
4. Subculturing:
a. Measure the OD600 of the overnight culture, dilute the culture tenfold, and measure OD600 again.
- Control: "2 ml YPD, 200 µl culture + 1800 µl YPD"
b. Transfer the overnight culture to 30 ml 2XYPD (+4% Glucose) medium such that the starting OD600 is 0.1-0.2.
- Volume of culture = 30ml * 0.1 / OD600
- Use: "45 ml 2XYPD + 5 ml 40% Glucose"
c. Incubate at 30°C, 250 rpm for 4-6 hours until OD600 reaches 0.6-0.8.
5. Preparation of Competent Cells:
NOTE: the following steps is for 10ml culture, adjust Buffer A volume according to the actual culture volume.
a. When OD600 reaches 0.6-0.8, centrifuge at 3000xg for 3 minutes, discard supernatant, and retain cells.
b. Resuspend cells in 5 ml Buffer A, centrifuge at 3000xg for 3 minutes, discard supernatant, and retain cells.
c. Resuspend cells in 400 µl Buffer A, distribute 100 µl per tube into 1.5 ml sterile tubes.
d. Add 5.5 µl DMSO to each tube and mix. Competent cells are ready for transformation.
6. Transformation ofPichia pastoris:
a. Mix 3 µg linearized plasmid DNA with carrier DNA, add to 100 µl competent cells. Total DNA volume should not exceed 20 µl.
- pGAPZaB-SVP/SVP-GNA plasmid digested with AvrII, recovered"
- "Carrier DNA thawed on ice, heated at 100°C for 15 minutes, placed on ice for 15 minutes before use"
b. Add 750 µl Buffer B, gently invert to mix.
c. Incubate at 30°C for 1 hour, gently invert every 15 minutes to mix.
d. Centrifuge at 2000xg for 10 minutes, discard supernatant, resuspend cells in 750 µl Buffer C.
e. Centrifuge at 2000xg for 10 minutes, discard supernatant, resuspend cells in 200 µl Buffer C.
f. Coat the suspension on YPD (+2% Glucose) plates with 100 µg/mL zeocin, incubate at 30°C for 3-4 days until colonies appear.
- Use: "47.5 ml YPD + 2.5 ml 40% Glucose + 50 µl 100 mg/mL zeocin"

All operations with potential risks of contaminating the culture should be conducted in a safety tablet.

1. Activation of Strain:
- Take 5 µl ofPichia pastoris SMD1168H glycerol stock stored at -80°C and streak on a YPD (+2% Glucose) plate. Incubate at 30°C for 2-4 days to activate the strain.
- Use: "47.5 ml YPD + 2.5 ml 40% Glucose"
2. Inoculation:
- Inoculate a fresh yeast colony into 5 ml YPD (+2% Glucose) liquid medium.
- Use: "47.5 ml YPD + 2.5 ml 40% Glucose"
3. Cultivation:
- Incubate at 28-30°C, 250 rpm overnight.
4. Subculturing:
a. Measure the OD600 of the overnight culture, dilute the culture tenfold, and measure OD600 again.
- Control: "2 ml YPD, 200 µl culture + 1800 µl YPD"
b. Transfer the overnight culture to 30 ml 2XYPD (+4% Glucose) medium such that the starting OD600 is 0.1-0.2.
- Volume of culture = 30ml * 0.1 / OD600
- Use: "45 ml 2XYPD + 5 ml 40% Glucose"
c. Incubate at 30°C, 250 rpm for 4-6 hours until OD600 reaches 0.6-0.8.
5. Preparation of Competent Cells:
NOTE: the following steps is for 10ml culture, adjust Buffer A volume according to the actual culture volume.
a. When OD600 reaches 0.6-0.8, centrifuge at 3000xg for 3 minutes, discard supernatant, and retain cells.
b. Resuspend cells in 5 ml Buffer A, centrifuge at 3000xg for 3 minutes, discard supernatant, and retain cells.
c. Resuspend cells in 400 µl Buffer A, distribute 100 µl per tube into 1.5 ml sterile tubes.
d. Add 5.5 µl DMSO to each tube and mix. Competent cells are ready for transformation.
6. Transformation ofPichia pastoris:
a. Mix 3 µg linearized plasmid DNA with carrier DNA, add to 100 µl competent cells. Total DNA volume should not exceed 20 µl.
- pGAPZaB-SVP/SVP-GNA plasmid digested with AvrII, recovered"
- "Carrier DNA thawed on ice, heated at 100°C for 15 minutes, placed on ice for 15 minutes before use"
b. Add 750 µl Buffer B, gently invert to mix.
c. Incubate at 30°C for 1 hour, gently invert every 15 minutes to mix.
d. Centrifuge at 2000xg for 10 minutes, discard supernatant, resuspend cells in 750 µl Buffer C.
e. Centrifuge at 2000xg for 10 minutes, discard supernatant, resuspend cells in 200 µl Buffer C.
f. Coat the suspension on YPD (+2% Glucose) plates with 100 µg/mL zeocin, incubate at 30°C for 3-4 days until colonies appear.
- Use: "47.5 ml YPD + 2.5 ml 40% Glucose + 50 µl 100 mg/mL zeocin"

All operations with potential risks of contaminating the culture should be conducted in a safety tablet.

1. Inoculation:
Inoculate a correctly sequenced fresh yeast colony into 5 ml YPG with 100 µg/mL zeocin (+4% Glycerol) liquid medium.
- Use: "46 ml YPG + 4 ml 50% Glycerol + 50 µl 100 mg/mL zeocin"
Incubate at 28-30°C, 250 rpm overnight.
2. Pre-cultivation:
Sterilize a 100 mL flask, add 30 mL YPG (+4% Glycerol) medium, inoculate with overnight culture, incubate at 30°C, 250 rpm for 14-16 hours until OD600 reaches 2-6.
- Use: "46 ml YPG + 4 ml 50% Glycerol"
3. Protein Fermentation:
Collect culture, centrifuge at 5000g for 5 minutes, discard supernatant, resuspend cells in 50 mL BMGY medium, adjust OD600 to approximately 1.0, continue in the 100 mL flask at 30°C, 250 rpm to induce expression.

Pichia pastoris Protein Collection
1. Collection of Supernatant:
Transfer fermentation broth to centrifuge tubes, balance, and centrifuge at 7000g, 4°C for 20 minutes. Carefully transfer supernatant to new tubes.
2. Concentration of Supernatant:
Use ultrafiltration to concentrate supernatant tenfold.
3. SDS-PAGE:
Prepare samples from concentrated supernatant for SDS-PAGE and run protein electrophoresis.

All operations with potential risks of contaminating the culture should be conducted in a safety cabinet.

Competent Cell Preparation and Transformation
1. Revival from cryopreservation
-Revival of -80°C stored via streaking on solid YPD(+2%Glucose)
- Culture in 30°C stationary incubator for 2 to 4 days for revival
2. Inoculation
-Inoculate a single yeast colony into 5ml liquid YPD(+2%Glucose)
3. Culture
-Overnight culture of yeast in shaking incubator at 28-30°C at 220RPM
4. Culture x2
-Using spectrometer, measure OD600 of the liquid culture, measure OD600 again for 10× diluted liquid culture (let control be 2ml LB)
-Transfer certain amount of liquid culture to 30ml 2×YPD(+4%Glucose) so that initial OD600 of the culture is between 0.2 and 0.3
-Incubate the culture in shaking incubator at 30°C and 250-300RPM for 4 to 6 hours, so OD600 reaches 0.8-1.0
5. Prepare competent cells
-Add according amount of liquid culture to 50ml centrifuge tubes based on number of tubes of competent cells you wish to prepare (5ml of liquid culture is needed to prepare one tube of competent cells)
-Centrifuge the liquid culture at 3000RPM and 20°C for 5 minutes, carefully remove the supernatant
-With half the volume (of originally removed liquid culture) of ddH2O, resuspend the pellet, centrifuge again at 3000RPM and 20°C for 5 minutes
-Repeat the previous step: with half the volume (of originally removed liquid culture) of ddH2O, resuspend the pellet, centrifuge again at 3000RPM and 20°C for 5 minutes
-With resuspend the washed pellet, distribute the resuspended pellet into 1.5ml sterilized centrifuge tube, 1ml per tube
-Centrifuge the 1.5ml tubes at 5000RPM and room temperature for 5 minutes, remove the supernatant to obtain the competent cells
6. Transformation
-Prepare LiAc Mix:


*For tubes of competent cells, expand the listed mix to Distribute the prepared LiAc mix into 1.5ml EP tubes
Prepare DNA Mix (50µl/tube, use to make up the volume)
DNA Mix 1 (Control 1): pCRCT+ddH2O
DNA Mix 2 (Control 2): Type9K+ddH2O
DNA Mix 3: pCRCT+DNA fragment for HR+ddH2O
*Total mass of plasmids (pCRCT/Type9K) should be around 250ng
*Total mass of DNA fragments for HR should be around 2000ng

*Per DNA fragment mass=
-Resuspend prepared competent cells with their according DNA mixes
-With 310μl LiAc Mix per tube, vortex and mix the competent cells
-Metal bath the transformation mix prepared in the previous steps at 42°C for 40 minutes
-Centrifuge the transformation at 5000RPM and room temperature for 2 minutes after metal bathing, carefully remove the supernatant, avoid pellet loss
-Via repetitive pipetting, resuspend the pellet with 500 μl sterilized water, after resuspension, spread the transformation mix onto SC-Ura (+2%Glucose plates)
-Incubate the plates at 30°C

MUST BE CONDUCTED WITHIN SAFETY CABINET
1. Colony PCR
Label the PCR tubes as accordingly; sterilize the PCR tubes by placing into clean bench with UV lights on
Add 10µl sterilized water to each of the PCR tube
Add yeast colonies to the PCR tubes; via repetitive pipetting, replicate the colonies onto labelled SC-Ura replica plate
Add 0.5µl 0.4M NaOH to every PCR tube; alkaline lysis at 95°C for 30 minutes in thermal cycler

Plasmid Elimination
1. Inoculation (In absence of light)
-Inoculate yeast colonies that shows correct colony PCR results into 5ml liquid YPD(+2%Glucose) with 1% 5-FOA
-Incubate in absence of light at 30°C and 200RPM for 24-48 hours in shaking incubator
2. Streaking (In absence of light)
-Streak with 10µl liquid culture onto YPD(+2%Glucose) solid
-Incubate at 30°C in stationary incubator for 2-3 days in absence of light
3. Streaking×2
-Inoculate single colonies onto a SC-Ura plate first (via streaking), without changing pipette tip, streak again on a YPD(+2%Glucose) plate
-Incubate at 30°C in stationary incubator for 2-3 days
4. Genome Sequencing
-Yeast that can not grow on SC-Ura have their plasmids completely eliminated, send the according yeast colonies on YPD plates for sequencing

Saccharomyces Culturing
5. Inoculation
-Inoculate a colony with correct sequencing results into 5ml liquid YPD(+2%Glucose)
-Incubate at 28°C to 30°C and 280RPM overnight
6. Pre-Culture
-Add 50ml liquid YPD(+2%Glucose) to sterilized 100ml conical flask
-Inoculate 500µl liquid culture into the conical flask, incubate at 30°C and 220 RPM at 1.5 to 2 hours so that OD600 reaches 0.4 to 0.6
7. Culture
Centrifuge pMVA+SCIE8 culture at 2000RPM for 5 minutes, carefully transfer the dodecane layer into yeast liquid culture, culture at 30°C and 220RPM overnight
8. GC-MS Analysis
-Transfer overnight liquid culture into a centrifuge tube
-Centrifuge at 12000RPM for 5 minutes
-Transfer and filter the layer of dodecane into GC-MS sample tube
-Mailing for analysis

Note: All operations must be conducted in a clean bench! Please ensure sterile techniques.
Day 1:
SCIE8 + pMVA Streaking:

• Take 5 µl of glycerol stock and streak on a CK dual-antibiotic LB agar plate.
• Incubate overnight at 37°C in an inverted position.

CK Dual-Antibiotic LB Plate: 50 ml LB + 35 µl C-antibiotic + 50 µl K-antibiotic

Day 2:
SCIE8 + pMVA Inoculation:

• Pick a single colony and inoculate into 5 ml CK dual-antibiotic LB medium.
• Incubate overnight at 37°C, 220 rpm.

CK Dual-Antibiotic LB Medium: 50 ml LB + 35 µl C-antibiotic + 50 µl K-antibiotic

Day 3:
SCIE8 + pMVA Expansion:

• Perform a 1:100 expansion of the culture into 100 ml CK dual-antibiotic 2YT medium.
• Incubate at 37°C, 220 rpm until OD600 reaches 0.6–0.8.
• Induce with 0.1 mM IPTG and add 1.5% glycerol as a carbon source.
• Incubate at 30°C, 200 rpm for 48 hours.

CK Dual-Antibiotic 2YT Medium: 50 ml 2YT + 35 µl C-antibiotic + 50 µl K-antibiotic
IPTG Addition (µl) = 100 × 1000 µl × 0.1 / 1000
Glycerol Addition (ml) = 100 × 1.5 / 50

Yeast Inoculation:

• Pick yeast colonies from the replica plate and inoculate into 5 ml YPD + 2% glucose medium.
• Incubate overnight at 30°C, 200 rpm.

YPD + 2% Glucose: 47.5 ml YPD + 2.5 ml 40% glucose

Day 4:
Yeast Expansion:

• Perform a 1:40 expansion of the overnight yeast culture into 100 ml YPD + 2% glucose medium.
• Incubate overnight at 30°C, 200 rpm.

YPD + 2% Glucose: 47.5 ml YPD + 2.5 ml 40% glucose

Day 5:
Co-culture of E. coli and S. cerevisiae
1. Dilute the yeast culture 10 times, measure the yeast culture OD600, and calculate the required amount of yeast culture.

• Control: 2000 µl YPD; Sample: 1800 µl YPD + 200 µl yeast culture
• Yeast amount = 100 ml × 40 / yeast OD600

2. Transfer the calculated amount of yeast culture to a centrifuge tube, centrifuge at 6000 g for 10 minutes, and carefully discard the supernatant.
3. Resuspend the yeast pellet in 48h cultured E. coli liquid, gently mix by pipetting, and incubate the mixture of E. coli and yeast at 28°C, 200 rpm for 48 hours.

---

Day 7:
1. Transfer the mixed culture of E. coli and yeast to a centrifuge tube, centrifuge at 12000 rpm for 5 minutes to collect the culture.
2. Add 5% dodecane to the collected culture, perform extraction, let it stand to allow separation, and then take the dodecane layer to filter into a GC-MS sample vial. Send for GC-MS analysis.

Reagent Preparation:

• LB:  1% tryptone, 0.5% yeast extract, 1% NaCl
• 2YT:  1.6% tryptone, 1% yeast extract, 0.5% NaCl
• YPD:  1% yeast extract, 2% peptone, add 2% d-glucose when used
• 50% Glycerol:  30 ml glycerol, 30 ml water

This protocol is written under the instructions of Prof. Huang.

The test was conducted according to the agricultural industry standard NY/T 1154.13-2008, using the leaf disc spray method at the National Key Laboratory of Green Pesticide, South China Agricultural University. The procedure is as follows:

1. Prepare clean, flat, appropriately sized kidney bean leaves with consistent leaf age.
2. Lay them flat in petri dishes that contain clean filter paper, with the underside facing up, and moisten the filter paper with water.
3. Use a brush to introduce 20 female adult two-spotted spider mites onto each leaf.
4. Use a sprayer to treat the leaves with female mites until the surface of the leaves and the mites' bodies are just covered with mist droplets. Allow them to stand in a natural environment until the droplets dry. Add a shallow layer of water around the leaves in the petri dish to prevent the mites from escaping, and cover the dish.
5. After treatment, place the test mites in an incubator with a temperature of (25±1)°C, humidity of about 60%, and a light cycle of L:D = (16:8) hours for rearing and observation.
6. After 72 hours, examine mortality by gently touching the insect bodies with a brush; those that do not move their legs or show any reaction are considered dead.

Each treatment is repeated three times, with controls treated with the same concentration of solvent and emulsifier.

For the GC-MS analysis, firstly, the exudates were briefly separated on a 30 m x 0.25 mm diameter capillary, with a 0.25 um film of HP-5MS UI from Agilent. Injection was then done in splitless mode at 250°C, the GC oven temperature was set at 50°C for 1 min followed by a linear gradient of 7°C/min up to 300°C and then 20°C/min to 330°C.

Helium was used as a carrier gas with a flow rate of 1 ml/min and molecules were ionized using electron ionization at 70 eV. The temperature of the transfer line was 200°C and that of the ion source 250°C. The MS data were recorded from 50 to 450 m/z.