Apparatus and Materials
Chemicals: H₂O, Ethanol, IPTG
Apparatus: Laminar flow hood
Resistance: Ampicillin, Chloramphenicol, Kanamycin

Procedure
1. Prepare the corresponding solutions according to the ratios in the following table:

Solution Type	Mass of Solute (g)	Solvent	Volume of Solvent (ml)
100mg/mL Ampicillin resistance	0.5	H₂O	5
50mg/mL Chloramphenicol resistance	0.5	100% Ethanol	10
50mg/mL Kanamycin resistance	0.5	H₂O	10
1M IPTG	2.383	H₂O	10

2. After complete dissolution, filter and sterilize in a laminar flow hood before aliquoting.
Store at -20°C.

Material
Chemicals: pfu, DNA Template, Forward Primer, Reverse Primer, pfu Buffer, dNTPs, ddH₂O
Apparatus: Vortex mixer, Desktop centrifuge, Thermocycler, 200 μl PCR tubes

Procedure
1. Prepare the total reaction mix based on the enzyme being used and total volume of PCR needed according to the table below:

Chemicals	Volume (μl)
pfu	1
DNA Template	1
Forward Primer	1
Reverse Primer	1
5 * Fastpfu Buffer	10
dNTPs	4
ddH₂O	32

2. Vortex to mix and then centrifuge.

3. Separate the total reaction mix into several equivalent aliquots 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.

Step	Temperature (℃)	Duration (s)	Note
1	98	120
2	94	30
3	56 (system-dependent)	30
4	72		Dependent on intended product length, pfu works at 15s/kb GOTO Step 2 ×30 times
5	4	∞	for short term storage

Material
Chemicals: Primers, pfu, pfu Buffer, dNTPs, ddH₂O
Apparatus: Thermocycler, vortex mixer, 200μl PCR tubes, centrifuge

Method
1. Pipette 2 μl of each of the prepared primers into a PCR tube to make a primer pool mix. Vortex to mix.

2. To another PCR tube, add the following with a micropipette:

Chemicals	Volume (μl)
Primer oligo mix	2
pfu	1
5 × Fastpfu Buffer	10
dNTPs	4
ddH₂O	33

3. Vortex and centrifuge

4. Set the temperature and time for PCR as shown in the table below

Step	Temperature (℃)	Duration (s)	Note
1	98	120
2	94	30
3	56	30
4	72		Dependent on intended product length, pfu works at 15s/kb GOTO Step 2 ×30 times
5	4	∞	for short term storage

5. Prepare another mixture according to the table below

Chemicals	volume/μl
PCR product from step 4	1
Forward primer (P-1)	1
Reverse primer (p-12)	1
pfu	1
5 × Fastpfu Buffer	10
dNTPs	4
ddH₂O	32

6. Vortex and centrifuge

7. Set the temperature and time for PCR as shown in the table below

Step	Temperature (℃)	Duration (s)	Note
1	98	120
2	94	30
3	56	30
4	72		Dependent on intended product length, pfu works at 15s/kb GOTO Step 2 ×30 times
5	4	∞	for short term storage

Materials
Chemicals: 10×H buffer, EcoRI, PstI, ddH₂O, DNA
Apparatus: Thermocycler, 200μl PCR tube

Method
1. Add the following to a PCR tube using a micropippette.

Chemicals	Volume (μl)
10× H buffer	10
EcoRI	2.5
PstI	2.5
DNA (vector or fragment)	5 μg
ddH₂O	Up to 100

2. Incubate the reaction at digestion temperature (37°C) for 2 hours in a thermocycler

3. Proceed to Gel electropherosis and Gel Extraction (refer to the respective protocols)

*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).

Materials
Chemicals: 10×T4 Ligase, T4 Ligation Buffer, DNA fragment, Vector, ddH₂O
Apparatus: Thermocycler, 200μL PCR tube

Method
1. Prepare a mixture in a PCR tube according to the table below:

Reagent	volume/μl
10× T4 Ligation buffer	2
T4 DNA ligase	1
DNA fragment	X
vector	Y
ddH₂O	Up to 20

\[ X = 3Y \times \left( \frac{C_{\text{Vector}} \times L_{\text{fragment}}}{C_{\text{Fragment}} \times L_{\text{vector}}} \right) \]

2. Incubate the reaction at digestion temperature (usually 37 °C) for 1 hour.

3. Recover the DNA using a purification kit (Refer to the DNA purification protocol)

4. Store the DNA sample at -20°C if not used immediately

Material

Chemicals	Apparatus
Buffer PE	Centrifuge tube (1.5 mL)
Buffer PW (ethanol added)	Pipette
ddH₂O	Centrifuge
	Razor blade
	Blue light transilluminator
	Spin column CA5
	Collection tube (2 mL)
	Microplate reader
	Balance
	Fridge (-20°C)

Procedure
1. Excise desired DNA band from agarose gel in blue light transilluminator using a razor blade and place it into a 1.5 mL centrifuge tube.

2. Measure the mass of the DNA band using a balance.

3. Add buffer PE in a volume equal to three times the mass of the excised gel (0.1 g=100μL); wait for 5-10 min to liquify the gel completely.

4. Transfer the gel solution into the spin column CA5 placed in a collection tube and wait for 2 min.

5. Centrifuge for 1 min at 12,000 rpm.

6. Discard the solution in the collection tube; add 600 μL buffer PW into the spin column CA5.

7. Centrifuge for 1 min at 12,000 rpm.

8. Repeat step 6-7.

9. Discard the solution in the collection tube.

10. Centrifuge for 2 min at 12,000 rpm.

11. Discard the solution in the collection tube.

12. Leave the spin column CA5 to dry at room temperature for ~5 min with the lid open; make sure no ethanol is left in the spin column CA5.

13. Discard the collection tube; transfer spin column CA5 to a centrifuge tube (1.5 mL).

14. Add 35 μL ddH2O into spin column CA5 on the membrane; centrifuge for 2 min at 12,000 rpm.

15. Transfer the solution in the collection tube to spin column CA5; centrifuge for 2 min at 12,000 rpm.

16. Discard spin column CA5.

17. Measure the concentration of the solution in centrifuge tube (1.5 mL) using microplate reader.

18. Store the remaining solution in -20°C fridge.

Material

Chemicals	Apparatus
Buffer P1	Centrifuge tube (1.5 mL)
Buffer P2	Pipette
Buffer P5	Centrifuge
Buffer PWT (ethanol added)	Vortex mixer
ddH₂O	Spin column CP3
Inoculated bacteria (minimum 4 mL)	Collection tube (2 mL)
	Microplate reader
	Fridge (-20°C)
	Fridge (4°C)
	Dry bath incubator

Procedure
1. Transfer 2 mL of the bacterial suspension into centrifuge tube (1.5 mL); centrifuge for 1 min at 12,000 rpm.

2. Discard the supernatant from the centrifuge tube and repeat step 1.

3. Add 150 μL buffer P1 into the centrifuge tube and resuspend the mixture with vortex mixer; the solution should appear turbid red.

4. Add 150 μL buffer P2 into the centrifuge tube; gently inverse the mixture until it is well mixed and turns a translucent lilac color.

5. Add 350 μL buffer P5 into the centrifuge tube; gently inverse the mixture until it is well mixed and turns a translucent yellow color. This step should be conducted within 2 min after the addition of buffer P2.

6. Centrifuge for 7 min at 12,000 rpm.

7. Transfer the supernatant into spin column CP3 placed in a collection tube; beware of resuspension of the pellet.

8. Centrifuge for 30 s at 12,000 rpm; discard the solution in the collection tube.

9. Centrifuge for 2 min at 12,000 rpm to remove the remaining ethanol.

10. Transfer spin column CP3 to a new centrifuge tube (1.5 mL); place the centrifuge tube in dry bath incubator set at 60°C with the lid open for 8 min.

11. Add 50μL ddH2O into spin column CP3; place the centrifuge tube in dry bath incubator set at 60°C with the lid closed for 5 min.

12. Centrifuge for 1 min at 12,000 rpm.

13. Discard spin column CP3; measure the concentration of the solution in centrifuge tube (1.5 mL) using microplate reader.

14. Store the remaining solution in -20°C fridge.

Apparatus and Materials

Chemicals	Apparatus
Agarose	Casting tray
TAE Buffer	well comb
Safe DNA Stain	voltage source
DNA Loading Buffer	Gel box
DNA Marker	Microwave
	UV light source

Method
Making a Agarose Gel:
1. Measure agarose powder and 1xTAE based on the size of the gel.

Size of Gel	Agarose (g)	1xTAE Buffer (mL)	Safe DNA Stain (µL)
Small	0.25	25	0.25
Medium	0.5	50	0.50
Large	1.0	100	1.00

2. Mix agarose powder with 1xTAE in a flask. Heat it until the solution boiled and the agarose inside completely dissolved. (could microwave in pulses, swirl the flask occasionally as the solution heats up)

3. With insulated gloves, let the gel cools down by rinsing the flask with water until there is no water vapor in the bottle mouth.

4. Drip Safe DNA Gel Stain to the solution. Swirl gently.

5. Pour the solution into a gel tray with well comb in it (there are different sizes of womb for different numbers of sample and experiment)

6. Wait for over 20 minutes at room temperature until the gel has completely solidified.

Loading Samples and Running the Gel:
7. Add 10x DNA Loading Buffer to each of the DNA samples if the enzyme mastermix used does not contain dye. Mix (1µL per 10µL, dilute 10 times)

8. Vortex the samples, then place PCR tubes in a centrifuge, making sure that all the samples are at the bottom of the tube.

9. Place the solidified gel into the gel box filled with 1xTAE

10. Carefully load 5µL of DNA marker into the first well, followed by 5µL of the samples added lane by lane.

11. Run the program at 140V for 20 minutes, until the dye line approaches about 60% of the other side of the gel.

12. Once the process ends, disconnect the electrodes, carefully remove the gel from the box.

13. Place the gel under UV light, visualize and analyze the DNA fragments.

Apparatus and Materials

Chemicals	Apparatus
Yeast extract	Pyrex bottle (500 mL)
NaCl	Electronic balance
Tryptone	Autoclave
H₂O	Sterile plate
Agar (required for agar plates)	Centrifuge tube (50 mL)
Antibiotics (A/C/K)	Clean bench
	Autoclave tape
	Microwave oven
	Fridge (4°C)

Procedure
Preparation of solutions
LB broth/agar

Chemicals	Mass/g
Yeast extract	4
NaCl	8
Tryptone	8
Agar (if producing agar)	6
ddH₂O	up to 400 mL

In Pyrex bottle (500 mL); swirl to mix.

Autoclaving
1. Loosen the cap of the Pyrex bottle; label it correctly with autoclave tape.

2. Sterilize by autoclaving at 121°C, 15 psi for 30 min; make sure to check the water level on the autoclave beforehand.

3. Allow the Pyrex bottle to cool to ~55°C. If not pouring plates immediately, store LB broth or LB agar at room temperature.

Pouring the plates (for LB agar)
1. Heat solidified LB agar in Pyrex bottles in microwave oven on low power until fully liquified; cool the Pyrex bottle to ~55°C. (Skip this step if directly proceeding from autoclaving)

2. Thaw antibiotic stock and mix well.
Note: The following procedure should be carried out in a clean bench using aseptic technique to prevent contamination.

3. Pour 50mL liquified LB broth in a centrifuge tube (50mL); add desired antibiotics and mix well.

Antibiotics	Volume/μL
Ampicillin (100 mg/mL)	75
Kanamycin (50 mg/mL)	50
Chloramphenicol (50 mg/mL)	35

4. Pour the mixture evenly on three empty plates; swirl the plate in a circular motion to distribute the media evenly on the plate.

5. Repeat step 3-4 to obtain more plates.

6. Allow the plates to solidify; flip them to prevent condensation forming on the agar.

7. Store the plates in a 4°C fridge.

Material

Chemicals	Apparatus
DNA Plasmid and Fragment	Thermocycler
T4 DNA Ligation Buffer	PCR Tubes
T4 DNA Ligase	Ice Bucket
BsaI	Microcentrifuge
ddH₂O

Method
1. Carry out the following actions in an ice bucket.

2. Calculate the amount of plasmid and fragments needed using the following formula:

\[ \frac{C_{\text{fragment}} \times V_{\text{fragment}}}{L_{\text{fragment}}} = 3 \times \frac{C_{\text{vector}} \times V_{\text{vector}}}{L_{\text{vector}}} \]

3. Add the designated volume of components into PCR tubes:

Chemicals	Volume (µL)
10 x T4 Ligation Buffer	2
DNA fragments	V fragment
Plamid	V vector
BsaI	1
T4 DNA ligase	1
ddH₂O	Up to 20 µL

4. Mix the mixture using vortex, and collect all the liquid at the bottom of the tube by centrifuging for a few seconds.

5. Place the tubes in the thermocycler and run the following process.

Step	Temperature (℃)	Duration (min)
1	37	2
2	16	2
3	Back to Step 1 (30 cycles)
4	37	10
5	60	5
6	10	∞

Apparatus and Materials

Chemicals	Apparatus
DNA fragments and vectors	Thermocycler
Gibson Buffer	Ice Box
ddH₂O	PCR tubes

Method
1. Carry out the following actions in an ice bucket.

2. Calculate the volume of vectors and inserts needed using the following formula:

\[ V_{\text{fragment}} = 3 V_{\text{vector}} \times \frac{C_{\text{vector}} \times L_{\text{fragment}}}{C_{\text{fragment}} \times L_{\text{vector}}} \]

3. Follow the following table, add all the components into a PCR tube.

Chemicals	Volume (µL)
Gibson Mix	5
DNA (Fragment)	X
Vector	Y
ddH₂O	Up to 10 µL

4. Mix the mixture using vortex, and collect all the liquid at the bottom of the tube by centrifuging for a few seconds.

5. Place the tubes in the thermocycler and run the following process.

Step	Temperature (℃)	Duration (min)
1	50	60

Material

Chemicals	Apparatus
LB agar plates with target bacteria	Thermal cycler
LB medium (required if testing transformants)	Pipette
Primers	Microcentrifuge tube
2X Flash master mix	Clean bench
ddH₂O	Other equipment required for gel electrophoresis
Agarose gel	Centrifuge
	Vortex mixer
	Incubator (37°)

Procedure
Preparation of materials
- Reaction mix

Reagents	Volume/μL
2X Flash master mix	15
Forward primer	1
Reverse primer	1
ddH₂O	13
Total	30

- Agarose gel
See "gel electrophoresis."

Colony PCR of Escherichia coli
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 in microcentrifuge tubes, with the quantity equivalent to the desired sample size.

3. Pick a single colony and dissolve in the reaction mix in a clean bench using aseptic conditions to prevent contamination.

4. (Only necessary during the first construction of a new plasmid transformant) Transfer 0.5 μL reaction mix onto the labelled replica plate in clean bench using aseptic conditions to prevent contamination; grow the plate overnight in 37°C incubator.

5. Place the microcentrifuge tubes with bacterial samples in thermocycler; PCR procedure will be set as the following program:

Step	Temperature/°C	Duration/s	Notes
Initial denaturation	98	90	/
Denaturation	94	10	/
Annealing	Tm-5	15	/
Extension	72	LR	GOTO Step 2 ×33 times
Final Extension	72	60	/
Preservation	4-10	/	/

*L = length of fragment, R = rate of polymerase

6. Run agarose gel electrophoresis on the PCR samples (verification, 5 μL/well)

7. Send the remaining PCR product with its primers for sequencing

Materials

Chemicals	Apparatus
BIOTECH SANGON DH5α E. coli competent cell	Electric dry bath
plasmid	Shaking incubator (37°C, 200 rpm)
LB plate with suitable antibody	Stationary incubator (37°C)
	Cell spreader
	1.5mL EP tubes

Method
1. Retrieve SANGON BIOTECH DH5α competent cells from the -80°C refrigerator and place it on ice

2. Add 30-50 μl to a 1.5 mL EP tube

3. Add 1μl the to-be transformed plasmid (or the entire gibson/goldengate assmbly product) to the EP tube containing the competent cells.

4. Mix gently by tapping on the EP tube. Let the mixture sit on ice for 30 minutes

5. heat at 42°C for 45s in a dry bath

6. Place the EP tubes back into ice for 2 minutes. Do not shake the tubes.

7. Add 700μl of sterile LB into the EP tubes in a clean bench. Mix thoroughly.

8. Incubate at 200 rpm for 1 hour in a 37°C shaking incubator

9. In a clean bench, pippette 100μl of the incubated cells onto a LB plate with the corresponding antibody and spread evenly with a cell spreader.

10. Out of the clean bench, centrifuge the contents in the tubes at 4500 rpm for 3.5min

11. In a clean bench, remove 500μl of the supernatent.

12. Aspirate to mix the remaining liquid thoroughly.

13. Pipette 100 μl of the remaining liquid onto a LB plate with the corresponding antibody and spread evenly with a cell spreader.

14. Remove the plates from the clean bench after they have dried.

15. Revert and place in a 37°C stationary incubator to incubate overnight.

Materials

Chemicals	Apparatus
E. coli competent cell	Electric dry bath
Plasmid	Shaking incubator (37°C, 200 rpm)
LB plate with suitable antibody	Stationary incubator (37°C)
	Cell spreader
	1.5 mL EP tube

Method
1. Retrieve E. coli competent cells from the -80°C refrigerator and place it on ice

2. Add 30-50 μl to a 1.5 mL EP tube

3. Add 1μl of the to-be-transformed plasmid (or all goldengate/gibson assembly products) to the EP tube containing the competent cells.

4. Mix gently by tapping on the EP tube. Let the mixture sit on ice for 30 minutes

5. heat at 42°C for 90s in a dry bath

6. Place the EP tubes back into ice for 5 minutes. Do not shake the tubes.

7. Add 300μl of sterile LB into the EP tubes in a clean bench. Mix thoroughly.

8. Incubate at 200 rpm for 30-60 min in a 37°C shaking incubator

9. In a clean bench, pippette 150-200 μl of the incubated cells onto a LB plate with the corresponding antibody and spread evenly with a cell spreader.

10. Remove the plates from the clean bench after they have dried.

11. Revert and place in a 37°C stationary incubator to incubate overnight.

Apparatus

• Sterile 15 mL culture tubes
• Shaking incubator

Materials

• Growth media (e.g., LB broth, YPD broth)
• Bacterial strain
• Antibiotic

Procedures
1. Add 4–5 mL of LB broth supplemented with the corresponding antibiotic into a sterile 15 mL culture tube.

2. Select a single colony and inoculate it into the culture tube.

3. Incubate overnight (16–20 hours) at 37 °C in a shaking incubator set to 220 rpm.

Materials

• Centrifuge (benchtop)
• Laboratory thermo-shaker
• Ultrasonic cell disruptor
• Ice
• Bacterial strain
• Appropriate antibiotics
• Inducer (IPTG)
• 20 mM Tris-HCl, pH 8.0

Method
Seed Culture
1. Prepare a sterile conical flask with LB broth (volume no more than 1/3 of the flask)

2. Inoculate the flask with the overnight pre-culture at a 1:100 dilution.

3. Grow the culture at 37 °C, shaking at 220 rpm, until the OD600 reaches 0.8–1.0.
  - As a quick visual check: tilt the flask at 45° and look downwards; if you can see the outline of your fingers through the liquid but cannot clearly distinguish fingerprint details, the OD600 is likely in the correct range.

*For terpenes:
Step 8: add glycerol to the final concentration of 1.5%

Induction of Protein Expression
4. Induce protein expression by adding IPTG to a final concentration of 0.3 mM.

  - Example: for a 50 mL culture, add 15 µL of 1 M IPTG stock solution.
  - General calculation formula:
  C1 V1 = C2 V2
    - C1 = initial IPTG concentration
    - C2 = target IPTG concentration (0.3 mM)
    - V2 = volume of culture
    - V1 = volume of IPTG stock to add

*For terpenes:
Step 10: add 10% of clean dodecane (sterilised by passing through a 0.22 µm syringe filter)

Main Culture
5. Continue incubation in a shaking incubator at a reduced temperature:

  - For overnight expression: 10–20 °C, shaking at 220 rpm for 16–20 hours.
  - For high-temperature induction: 30–37 °C, shaking at 220 rpm for 1–4 hours.

*For terpenes:
Step 11: keep incubation to 2~3 days

Materials
Buffers and solutions:
- Buffer PB, PW (TIANGEN)
- ddH₂O

Enzymes and buffers:
- 10 * H buffer, Nhe I (Takara Bio)

Nucleic acids and oligos:
- Purified plasmid

Centrifuges:
- Benchtop centrifuge; able to accomodate 2ml EP tubes

Special equipment:
- 37°C dry bath incubator
- CB2 spin column, collection tube (TIANGEN)

Method
Linearization
1. Plasmids are extracted from overnight E. coli culture with TIANprep Midi Plasmid Kit (TIANGEN) and have concentration quantified with plate reader

2. With micropipette, add the following to a 1.5ml EP tube

3. Vortex and centrifuge to homogenate the mix if necessary

4. Place the EP onto a dry bath incubator(37 degree celcius), wait for 30 minutes

Purification
5. Add 500μl of buffer PB (TIANquick Midi Purification Kit, TIANGEN) to the reaction mix from step 4

6. Vortex to mix, centrifuge if necessary

7. Add 500μl buffer BL to a CB2 spin column, assembled with a collection tube

8. Centrifuge the CB2 spin column at 1200RPM for 1 minute, discard liquid in the collection tube, place back the collection tube

9. To each spin column, add 600μl of liquid from step 6, leave to stand for 2 minutes at room temperature

10. Centrifuge spin columns at 12000RPM for 1 minute

11. Add back liquid from collection tube to the spin column with a micropipette, place back the collection tube, leave to stand for 2 minutes at room temperature

12. Centrifuge spin columns at 12000RPM for 1 minute

13. Repeat steps 11 and 12

14. Discard liquid in the collection tube, add 600μl buffer PW to spin column, place back the collection tube

15. Centrifuge spin columns at 12000RPM for 1 minute

16. Repeat steps 14 and 15

17. Discard liquid in the collection tube, place back the collection tube, centrifuge spin columns at 12000RPM for 2 minutes to remove buffer PW

18. Discard the collection tube, place spin column onto corresponding, labelled 1.5ml EP tubes

19. Open the lids of spin columns, place spin columns onto dry bath incubator set to 60°C, wait for 3 minutes

20. Add 30μl ddH₂O to the center of the spin column, with pipette tip not in contact with the white material

21. Close the lid of spin columns, place on 60°C dry bath incubator for 5 minutes to dissolve DNA

22. Centrifuge the spin columns at 12000RPM for 2 minutes

23. With micropipette, add back liquid in the collection tube into the spin column

24. Repeat steps 21 and 22

25. Discard spin columns, store EP tubes at -20°C after measuring and labelling the DNA concentration with plate reader

Materials
Cells:
- 50ml P. pastoris culture, OD600 1.2-1.5

Buffers and solutions:
- 1M sorbitol solution, pre-cooled at 4°C

Centrifuges:
- Refrigerated tabletop highspeed centrifuge; able to accomodate 50ml centrifuge tubes, centrifuge at 5000RPM and 4°C

Media:
- MD, solid

Method
1. In clean bench, divide 50ml culture yeast culture (from extended inoculation, see P. pastoris extended inoculation protocol), OD600 at 1.2 to 1.5, into two 50ml centrifuge tubes equally; swirl the culture to resuspend yeast pellet if necessary before dividing

2. Centrifuge divided cultures at 4°C, 5000RPM for 5 minutes

3. In clean bench, gently pour away supernatant, avoid pouring away any precipitated yeast

4. Resuspend the precipitated pellets with 25ml sterilized water each; invert and aspirate with pipette very gently if necessary

5. Centrifuge at 4°C, 5000RPM for 5 minutes

6. In clean bench, gently pour away supernatant, avoid pouring away any precipitated yeast

7. Resuspend the precipitated pellets with 5ml pre-cooled 1M sorbitol each; invert and aspirate with pipette very gently if necessary

8. Decant content of one centrifuge tube into the other

9. Centrifuge at 4°C, 5000RPM for 5 minutes

10. In clean bench, gently pour away supernatant, avoid pouring away any precipitated yeast

11. Resuspend remaining pellet with 1ml pre-cooled 1M sorbitol

12. Divide the resuspended solution into sterilized 2ml EP tubes, 200μl each; store at -80°C if the prepared competent cells are not used immediately

Materials
Cells:
- Prepared P. pastoris competent cells

Buffers and solutions:
- Absolute ethanol
- 1M sorbitol solution, pre-cooled at 4°C and at room temperature

Nucleic acids and oligos:
- Purified linearized plasmid

Centrifuges:
- Benchtop centrifuge; able to accomodate 2ml EP tubes
- Benchtop centrifuge; able to accomodate 2ml EP tubes

Special equipment:
- Electroporation system
- Electroporation cuvettes, pre-cooled during transformation
- Shaking incubator (30°C, 220RPM)
- Stationary incubator (30°C)

Media:
- MD plates

Method
Note: All reagents used in this protocol except from absolute ethanol (i.e. 1M sorbitol) should be sterilized beforehand; it is advised to bring in a conical flask for waste liquid for electroporation cuvette preparation into clean bench

Preparation of electorporation cuvettes
1. In a clean bench, add absolute ethanol to a cuvette to almost full, invert gently several times to wash

2. Discard liquid in the cuvette

3. Repeat steps 1 and 2 twice

4. Add 1M sorbitol to the cuvette to almost full, invert gently several times to wash

5. Discard liquid in the cuvette

6. Repeat steps 4 and 5 twice

7. Place the cuvette lids’ inner side facing upwards, dry cuvettes and lids for 1hr with clean bench UV light and ventilation on

8. Store prepared cuvettes in a sealable plastic bag at -20°C

Electrotransformation
9. In a clean bench, into each pre-cooled cuvette, add 200μl prepared P. pastoris competent cells each, label the cuvette lids for better identification, place cuvettes into ice bath

10. Into each cuvette, add 20μl of desired linearized plasmids; total mass of DNA added should be between 5 to10μg; do not aspirate; too large a volume of DNA added in this step will reduce transformation efficiency, close the cuvette lids

11. Ice bath the cuvettes outside clean bench for 15 minutes

12. Electroporate at 1500V, 400Ω, 25μF with the electroporation system (Bio-Rad Gene Pulsar); adjust cuvette diameter accordingly (2mm)

13. Add 1ml pre-cooled 1M sorbitol into each cuvette in a clean bench immediately after electroporation

14. Aspirate and transfer all liquid within the cuvette to a sterilized 1.5ml EP tube with a micropipette

15. Incubate the EP tube in a shaking incubator (30°C, 220RPM) for 2h

16. Centrifuge EP tubes at 5000RPM for 5 minutes, remove and discard 1ml supernatant with a micropipette in a clean bench

17. Resuspend the remaining pellet by aspiration, inoculate all the remaining liquid onto an MD plate (see pastoris culture media preparation protocol) with spread plate method using a cell spreader

18. Incubate the plate at stationary incubator set to 30°C, incubate for 2-3 days for visible colonies

Materials Buffers and solutions:
- Distilled water
- Deionized water

Special equipment:
- Autoclave
- Syringe, syringe filter

Chemicals (reagent grade unless otherwise stated):
- Sorbitol
- D-glucose
- YNB (yeast nitrogenous base)
- Biotin
- G418 (geneticin)
- Glycerol
- Methanol
- K₂HPO₄·3H₂O
- KH₂PO₄
- NaOH

Method
1. Prepare the following reagents/solutions, scale up/down total volume and amounts of each reagent as appropriate (unless otherwise stated, store at room temperature):

Reagent	Method
1M Sorbitol Solution	Dissolve 91.09g sorbitol in distilled water to exactly 500ml total volume, sterilize with autoclave at 121°C, 20 minutes, store at 4°C
10× Glucose Solution	Dissolve 200g D-glucose into 1000ml distilled water, sterilize with autoclave at 121°C for 15 minutes or with sterile filtration
10×YNB Solution	Dissolve 134g YNB (yeast nitrogenous base) into 1000ml distilled water, filter to sterilize, heat with microwave oven until YNB fully dissolves, store at 4°C
500× Biotin Solution	Dissolve 20mg biotin into 100ml distilled water, filter to sterilize, store at 4°C
200mg/ml G418 Solution	Dissolve 1g G418 in distilled water to exactly 5ml total volume, filter to sterilize, store at -20°C
10× Glycerol Solution	Add deionized water to 100g glycerol to exactly 1000ml total volume, sterilize with autoclave at 121°C for 20 minutes, store at 4°C
10× MeOH Solution	Add deionized water to 50ml methanol to exactly 1000ml total volume, filter to sterilize, store at 4°C
1M Potassium Phosphate Buffer (PH6.0)	Add deionized water to 3.15g K₂HPO₄·3H₂O (0.01381M), 11.73g KH₂PO₄ (0.08619M) to exactly 1000ml total volume, sterilize with autoclave at 121°C for 20 minutes, store at 4°C
400mM NaOH	Dissolve 0.8g NaOH into 50ml distilled water

2. Prepare the following media:

Media	Method
MD (1L)	Sterilize 800ml water with autoclave, at 60 degrees, add 100ml 10×YNB, 2ml 500×Biotin, 100ml 10×D-glucose If preparing solid media, add 15g agar before sterilizing water
BMGY/BMMY (1L)	Dissolve 10g yeast extract and 20g tryptone into 700 distilled waterSterilize with autoclave at 121°C for 20 minutesCool to room temperature, add the following mix: 1. 100ml 1M potassium phosphate buffer (PH6.0) 2. 2ml 500× biotin 3. 100ml 10× glycerol When preparing BMMY, use 100ml 10× MeOH instead of 10× glycerol
YPD (1L)	Add 10g yeast extract, 20g tryptone to 900ml distilled water Add 20g agar if preparing solid mediaSterilize with autoclave at 121°C for 20 minutes Add 40% glucose solution to 2% total glucose concentration after sterilization when using the prepared media Add appropriate amounts of 100mh/ml zeocin for zeocin-containing media

Note: stir with stirring rod or invert flasks to mix when appropriate

Materials
Cells:
- P. pastoris glycerol stock

Buffers and solutions:
- Absolute ethanol
- 1M sorbitol solution, pre-cooled at 4°C and at room temperature

Centrifuges:
- Benchtop centrifuge; able to accomodate 2ml EP tubes
- Benchtop centrifuge; able to accomodate 2ml EP tubes

Special equipment:
- Shaking incubator (30°C, 220RPM)
- Stationary incubator (30°C)
- Cell density meter

Media:
- YPD+2% glucose, liquid

Method
Note: Per 50ml final, extended pastoris culture, roughly ~6 preps of competent cells could be prepared

1. In clean bench, inoculate 5μl glycerol stock of desired yeast strain to YPD+2% glucose plate with streak plate method using micropipette

2. Incubate the plate in 30°C stationary incubator for 2 days for visible colonies

3. In clean bench, inoculate a very large amount of yeast (~roughly a ball 4mm in diameter on pipette tip) into 5ml YPD+2% glucose with a micropipette; do not need to pick up single colonies as the culture is homogenous

4. In shaking incubator (30°C, 220RPM), incubate the 5ml culture overnight

5. Measure OD600 of the 5ml culture, dilute by 10× if necessary; operations must be conducted in clean bench whenever culture needs to be opened

6. Inoculate the 5ml culture to 50ml YPD+2% glucose so that starting OD600 of the 50ml culture is between 0.2 and 0.5

7. Incubate the 50ml culture in shaking incubator (30°C, 220RPM), proceed to competent cell preparation when OD600 reach 1.2-1.5

Materials
Cells:
- P. pastoris transformant

Buffers and solutions:
- Sterilized water

Media:
- YPD+2% glucose+1mg/ml zeocin, solid

Special equipment:
- Cell spreader
- Stationary incubator (30°C)

Method
1. In clean bench, to the MD plate with pastoris transformants, add 3ml sterilized water

2. Resuspend the colonies with cell spreader, aspirate with micropipette if necessary

3. With micropipette, transfer the resuspended transformants to a 15ml centrifuge tube

4. Vortex to homogenate the suspension

5. In clean bench, add 100μl transformant suspension to 10ml sterilized water, invert to mix

6. Inoculate the diluted suspension to YPD+2% glucose+1mg/ml zeocin plates with cell spreader

7. Incubate the plates in a stationary incubator (30°C) for 3-4 days for visible colonies

Materials
Cells:
- High-copy transformants

Buffers and solutions:
- 400mM NaOH solution
- Sterilized water
- 2× Flash buffer (CoWin Biosciences)

Nucleic acid and oligos:
- Colony PCR primers, forward and reverse (10pmol/μl)

Centrifuges:
- Low-speed benchtop centrifuge, capable of holding PCR tubes

Media:
- YPD+2% glucose+2mg/ml zeocin, solid

Gel:
- Agarose gel, 16 wells, scale up with number of colonies

Special equipment:
- Stationary incubator (30°C)

Method
1. Label eight 8 PCR tube strips 1 through 16 and A to D (i.e. A1, A2...D15, D16, a total of 64 tubes)

2. Sterilize the A strip tubes (i.e. A1, A2...A15, A16) with lids off in clean bench with UV lights turned on for 30 minutes

3. In clean bench, add 20μl sterilized water to each of the A strip tubes

4. Out of clean bench, add 19μl of the colony PCR mix (recipe below) to each of the D strip tubes; it is advisable to prepare a 18×(in volume; scaled 18× for 16 tubes, scale up as needed) mix before distributing to each tube; store the PCR mix at 4°C if not to be used immediately

Reagent	Volume (1×)/μl	Volume (18×)/μl
2× Flash buffer	10	180
ddH₂O	7.4	133
Forward primer	0.8	14.5
Reverse primer	0.8	14.5

5. To each B strip tube, add 10μl 40mM NaOH solution (5ml 400mM NaOH solution+45ml distilled water, invert to mix)

6. Prepare a replica plate (YPD+2% glucose+2mg/ml zeocin, solid) with squares labeled 1 to 16

7. In clean bench, remove a colony from selection plate with micropipette, aspirate in tube A1, inoculate 2.5μl tube mixture to square 1 of the replica plate

8. Transfer 10μl of tube A1's contents to tube B1

9. Repeat step 7 and 8 fifteen times, with a different colonies each time, to respective tubes/squares

10. Out of clean bench, vortex to homogenate strip B tube contents, centrifuge if needed;incubate replica plate in 30°C stationary incubator

11. With thermocycler, lyse yeast cells with alkaline lysis by incubating B strip tubes at 95°C for 20 minutes

12. Centrifuge B strip tubes at 4500 RPM with low-speed benchtop centrifuge for 5 minutes

13. Transfer supernatant of B strip tubes to their corresponding C strip tubes

14. Transfer 1μl of content of each C strip tube to their corresponding D strip tube, PCR to amplify the DNA (see PCR protocol; Flash amplifies DNA at 15s/kb)

15. Proceed to gel electrophoresis with colony PCR products (see DNA agarose gel electrophoresis protocol)

16. Amplify tubes with positive electrophoresis results with their C tube contents using a 50μl PCR mix; PCR products shall proceed for sequencing:

Reagent	Volume (1×)/μl
2× Flash buffer	25
ddH₂O	23
Forward primer	1
Reverse primer	1
Lysate supernatant (C tube)	1

Materials
Chemicals:
- 20mM Tris-HCl Buffer pH8.0
- Induced bacterial culture

Apparatus:
- Vortex Mixer
- Ice Bath
- Cell Sonicator

Procedures:
1. Harvest the cell pellet by centrifugation at 5,000 rpm for 10–15 min at 4 °C, using a 50 mL centrifuge tube.

2. Resuspend the pellet in an appropriate volume of Tris-HCl lysis buffer, ensuring the pellet is completely covered (typically 1 mL buffer per 100 mg cells).

3. Vortex mixes the cell pellet with the buffer thoroughly.

4. If sonication will not be performed immediately, store the resuspended cells at –20 °C

5. Prepare an ice–water mixture in a beaker. Keep the sample tubes immersed in the ice–water bath throughout the sonication to maintain a low temperature.

6. Insert the sonicator probe into the sample, keeping it ~1 cm above the bottom of the tube and avoiding direct contact with the walls or bottom.

7. Sonicator settings:
- Power: 60 W
- Frequency: 25 kHz
- Pulse: 3 s on, 6–10 s off (to prevent overheating)

8. Sonicate for 3–5 min. Excessive heat generated during sonication can denature proteins if cooling is not maintained.

9. Completion of sonication is indicated by a clear, foam-free lysate.

10. Centrifuge the sonicated samples at 12,000 rpm for 7–15 min at 4 °C.

11. Carefully separate the supernatant (s) and pellet (p) into 50 mL centrifuge tubes, and label them clearly.

12. Resuspend the pellet at an equal volume of 20 mM Tris-HCl buffer (pH 8.0) relative to the collected supernatant.

13. After each use, clean the sonicator probe thoroughly with distilled water to avoid protein residues and cross-contamination.

Chemicals:
- PAGE Gel Fast Preparation Kit (10%) [Epizyme Biotech, China]
  - Upper gel solution
  - Upper gel buffer
  - Lower gel solution
  - Lower gel buffer
- 5× SDS loading buffer
- Prestained protein marker (10–200 kDa)
- 1× Tris-Glycine SDS-PAGE running buffer
- Fast SDS-PAGE staining buffer (Feto)
- Protein Samples

Apparatus:
- Spacer plate
- Short plate (glass)
- Casting frame
- Gel cassette assembly
- Casting stand
- Gel cassette sandwich (gel cassette “sandwich” structure)
- Electrode assembly
- Companion assembly (auxiliary component)
- Mini tank and lid (electrophoresis chamber)
- Buffer dam
- Power supply
- Vortex mixer

Procedure:
Step 0: Preparation of 1× Running Buffer
1. Mix 100 mL of 10× Tris-Glycine-SDS Running Buffer with 900 mL of distilled water.

2. Transfer into a clean bottle, label as “1× Running Buffer (freshly prepared),” and mix well.

Step 1: Gel Cassette Assembly
3. Clean and dry the short plate and spacer plate.

4. Place the short plate on top of the spacer plate to form a gel cassette. Apply gentle pressure and secure it in the casting frame.

5. Fill the gap between the plates with tap water and leave for 5 min to check for leakage.

6. If the water level remains stable, discard the water and proceed to gel preparation.

Step 2: Resolving and Stacking Gel Preparation
7. Prepare resolving gel solution according to the desired gel percentage.

Thickness(mm)	0.75	1.0
Upper gel solution + Upper gel buffer(mL)	0.5 each	0.75 each
Lower gel solution + Lower gel buffer(mL)	2.0 each	2.7 each

8. Just before pouring gel solution into gel cassette, add the appropriate amount of polymerization initiators (APS and TEMED) according to the ratio 1:100.

9. Using a 1000 µL pipette, load the resolving gel solution into the cassette until the height is about 1 cm below the bottom of the comb. Carefully overlay with 100% ethanol to prevent air exposure.

10. Allow 15–30 min for polymerization. A slight drop in gel height is normal.

11. Remove the ethanol and add the stacking gel solution with initiators. Insert the comb gently.

12. Allow another 15–30 min for the stacking gel to polymerize.

If the assembled gel is not used immediately, seal the bottom with Parafilm, place the cassette in a sealed bag, and submerge it completely in 1× Running Buffer. Store at 4 °C.

Step 3: Sample Preparation
13. Collect the supernatant and pellet fractions after sonication. Label PCR tubes accordingly.

14. Add 16 µL of sample and 4 µL of 5× loading buffer to each tube.

15. Vortex briefly and centrifuge to mix and pellet.

16. Heat the samples at 100 °C for 6–10 min in a PCR thermal cycler using the “protein” program.

Step 4: Gel Setup
17. Place the gel cassette with its holder into the electrophoresis tank.

18. Fill the inner chamber with freshly prepared 1× Running Buffer until the wells are completely covered. Wait 5–10 min to ensure there is no leakage.

19. Fill the outer chamber with 1× Running Buffer. Note: inner buffer must be fresh, while outer buffer can be reused.

Step 5: Sample Loading and Electrophoresis
20. Thaw the prestained protein marker in advance. Load 6 µL into the designated lane.

21. Load 6–10 µL of each sample into the wells, following the recorded sample order. Avoid introducing bubbles.

22. Connect the power supply and run at 150 V. Electrophoresis typically lasts 45–50 min, depending on gel concentration and voltage.

23. Stop running when the dye front approaches the bottom of the gel. Over-running may cause the marker to run off the gel.

Step 6: Staining and Destaining
24. Remove the gel carefully and place it in a staining tray.

25. Add sufficient Fast SDS-PAGE Staining Buffer to cover the gel. Place the tray on an orbital shaker for 5–10 min.

26. Discard the staining solution into a waste container and rinse the gel with distilled water. Shake gently for destaining until the background is clear.

Step 7: Result Analysis
27. Transfer the gel onto a white background plate and place it in the gel documentation system.

28. Compare the sample bands with the protein marker to estimate molecular weights.

29. Capture and save images for record-keeping.

Buffer formula: (for reference only)

pH	1M Citric Acid/ml	1M Sodium citrate/ml
3	0.930	0.09
4	0.655	0.360
5	0.410	0.605
6	0.190	0.87

pH	Tris-HCL/ml	0.1M NaOH/ml
7	49.20	0.80
8	45.35	4.65

Apparatus and Materials
Chemicals:
- 20mM tris buffer
- Buffer with desired pH
- 200mM imidazole tris buffer
- 20mM imidazole buffer (with desired pH)
- 200mM imidazole buffer (with desired pH)
- Ni-TED beads
- 20% ethanol
- Coomassie blue G-250 dye

Apparatus:
- Clamp and stand
- Syringe barrel (or other barrel like stuff, better with a flow regulator)
- 50/15ml centrifuge tubes (acc to the volume of the supernatant)
- Filter plate
- 96-well plate

Method:
1. Assemble the Ni-TED affinity column: (note: the system may be available already)
  1. Prepare a syringe and dispose of the needle. Take out the plunger.
  2. Place a filter plate at the bottom of the syringe barrel
  3. Prepare IV infusion set and take the roller clamp. Snip off a short section of the tube and connect it to the syringe. Put the section through the flow regulator. Make sure it's air tight.
  4. Close the flow regulator to seal
  5. Clamp the system steadily onto the stand
  6. Get His-tag Ni-TED beads (His-Tag Protein Purification Agarose Magnetic Beads) from 4°C (Note: the beads can be reused so they may be available already)
  7. Shake the beads gently to allow it to mix with 20% ethanol.
  8. Add beads to the the syringe barrel (cut the tip wider to allow smoother pull)
    1. Ni-TED beads typically bind >10 mg of His-tagged protein per mL of beads for a protein of ~60 kDa. Usually add 2 - 3 ml

2. Open the flow regulator to discard the ethanol into the waste container

3. Wash the beads with 200mM imidazole tris buffer (volumn should be greater than the volume of the beads)

4. Wash the beads a few times with the desired buffer to clean any residual imidazole
  1. Can test the pH of the fluid flown out (imidazole is basic)

5. After all fluid is flown out, close the flow regulator to seal the tube.

6. Pour the supernatant in. Gently stir to allow efficient binding

7. Wait for 10~30 min depending on the sample size to allow binding.
  1. Reloading may be performed for large sample sizes with small amount of beads.

8. Label a 15ml centrifuge tube: FT (flowthrough) with the protein names and dates. (use the same tube for the secondary binding if there is)

9. Slowly drip the sample into the labelled tube.
  1. Note: binding can also happen with the supernatant slowly flowing through

10. After dripping stops, add 20mM imidazole buffer with desired pH to wash a few times, getting rid of any residual impurities
  1. Store the first 10ml of the wash into a 15ml centrifuge tube labelled W1 (waste 1)

11. Close the flow regulator and add 5ml of 200mM imidazole solution with desired pH (formulation: buffer + imidazole)

12. Leave to stand for 10~30 min for adsorption of imidazole (stir to allow binding)

13. Use a 15ml centrifuge tube to get the 5ml of eluate. Label the tube E or Elu

14. Open the flow regulator to collect the imidazole solution (allow slow drip)

15. Test with Coomassie blue G-250 dye from the tip of the flow regulator to see if still a large amount of protein present
  1. If a drastic color change to blue is observed, add 1ml of 200mM imidazole again to further collect the eluate. Repeat till the color change is small)

16. Proceed to sample preparation and SDS-PAGE for evaluation

17. Store the eluate at 4°C
  1. If ultrafiltration / dialysis can not be done on time, store the sample at -20

Column Material Recycling:
1. Wash with 500mM imidazole tris buffer to get rid of any bound proteins

2. Wash with 20mM Tris buffer to wash away any residual imidazole

3. Fill with 20% ethanol and store in the refrigerator at 4°C.

Chemicals:
- Protein sample after purification
- Pure buffer with desired pH
- 0.1M NaOH solution
- 20% ethanol

Apparatus:
- Ultrafiltration tube with correct membrane size
  - If 10,000 inscribed, then the protein with Mr greater than 10kDa cannot flow through
- 15ml centrifuge tube

Method:
1. Ultrafiltration tube preparation:
  1. Pour 0.1 NaOH solution into the tube, allowing the filter membrane to fully immerse in the solution
  2. Wait for 10 min
  3. After 10 min, pour out the NaOH solution and wash with tab water to wash away any residual NaOH (may test with pH indicator)

2. Pour the sample into the upper section.

3. Close the tube tightly. Centrifuge at 4°C 5500 rpm for 25 min

4. After 25 min, take the membrane section out of the tube and dispose of the fluid at the bottom of the tube (which contains filtered imidazole)

5. Put the membrane section back and fill the membrane section with a large amount of buffer (with desired pH)

6. Repeat centrifugation at least 4 times

7. To collect the final solution, pipette up and down in the membrane section and pour the ultrafiltered sample into a 15ml centrifuge tube. Store the sample at 4 °C or add 50% glycerol and store at -20°C

8. Ultrafiltration tube recovery:
  1. Wash the membrane with tab water and then immerse with NaOH for 10min
  2. Wash the membrane again and immerse with 20% ethanol
  3. Store at 4 °C =======

Chemicals	Apparatus
Protein sample after purification	Ultrafiltration tube with correct membrane size   - If 10,000 inscribed, then the protein with Mr greater than 10kDa cannot flow through
Pure buffer with desired pH	15ml centrifuge tube
0.1M NaOH solution
20% ethanol

Procedure:
1. Ultrafiltration tube preparation:
  1. Pour 0.1 NaOH solution into the tube, allowing the filter membrane to fully immerse in the solution
  2. Wait for 10 min
  3. After 10 min, pour out the NaOH solution and wash with tab water to wash away any residual NaOH (may test with pH indicator)

2. Pour the sample into the upper section.

3. Close the tube tightly. Centrifuge at 4°C 5500 rpm for 25 min

4. After 25 min, take the membrane section out of the tube and dispose of the fluid at the bottom of the tube (which contains filtered imidazole)

5. Put the membrane section back and fill the membrane section with a large amount of buffer (with desired pH)

6. Repeat centrifugation at least 4 times

7. To collect the final solution, pipette up and down in the membrane section and pour the ultrafiltered sample into a 15ml centrifuge tube. Store the sample at 4 °C or add 50% glycerol and store at -20°C

8. Ultrafiltration tube recovery:
  1. Wash the membrane with tab water and then immerse with NaOH for 10min
  2. Wash the membrane again and immerse with 20% ethanol
  3. Store at 4 °C >>>>>>> bbc171f876e9a44a1e45660d5a659a79f956ad4c

Apparatus and materials
Chemicals:
- Pure water
- Protein sample after purification

Apparatus:
- Dialysis bag (with correct pore size)
- 250 ml beaker (for heating and dialysis, can use a bigger one for dialysis if overnight)
- Clamps for dialysis bag

Procedure:
1. Immerse the dialysis bag in pure water in the 250 ml beaker for heating

2. Heat the dialysis bag in microwave heater until boiling

3. Cool the dialysis bag and clamp the dialysis bag tight at one end
  1. Check for leak using pure water

4. Pour the sample into the bag and clamp tight at the other end

5. Fill the beaker for dialysis with the according buffer solution and put the dialysis bag in

6. Wait for 30-60 min to allow dialysis

7. Pour the water in the beaker away and refill the beaker with pure water

8. Repeat at least 3 times

9. Collect the solution in the bag and store at -20 =======

Chemicals	Apparatus
Pure water	Dialysis bag (with correct pore size)
Protein sample after purification	250 ml beaker (for heating and dialysis, can use a bigger one for dialysis if overnight)r
	Clamps for dialysis bag

>>>>>>> bbc171f876e9a44a1e45660d5a659a79f956ad4c

Apparatus and Materials

Materials	Apparatus
DNS reagent	Thermal Cycler
Glucose solution (50 mg/μL)	Vortex Mixer
1% chitin solution	Centrifuge
	Spectrophotometer

Procedure
1. Prepare 6 tubes of solutions according to the data in the table below (use an 8-well PCR strip and label them from 0 to 5).

PCR Tube Number	[C] (Concentration)	Glu / μL	1% Chitin Solution / μL
0	0	0	50
1	0.2	2	48
2	0.4	4	46
3	0.6	6	44
4	0.8	8	42
5	1.0	10	40

2. Add 100 μL of DNS reagent to each PCR tube.

3. Vortex for 5 seconds to mix thoroughly.

4. Centrifuge for 10 seconds to ensure no liquid adheres to the tube walls.

5. Place all tubes in the Thermal Cycler, set the temperature to 100 °C, and heat for 2 minutes.

6. Remove the tubes from the Thermal Cycler.

7. Take 6 1.5 mL centrifuge tubes and label them from 0 to 5.

8. Add 450 μL of water to each 1.5 mL centrifuge tube (3 times the volume of the liquid in the PCR tubes).

9. Transfer 150 μL of the solution from each PCR tube to the corresponding 1.5 mL centrifuge tube (match the tube numbers).

10. Vortex for 5 seconds to mix thoroughly.

11. Starting from Tube 0, use a pipette to aspirate 200 μL of the mixed solution from each 1.5 mL centrifuge tube and add it to the corresponding well of a 96-well plate (Tube 0 → Well A, Tube 1 → Well B, Tube 2 → Well C, Tube 3 → Well D, Tube 4 → Well E, Tube 5 → Well F).

12. Measure the absorbance using a Spectrophotometer at a wavelength of 540 nm.

13. Plot a graph with absorbance on the y-axis and glucose concentration on the x-axis, then draw a line of best fit.
  - If the coefficient of determination (R²) is greater than 0.99, the standard curve is usable.
  - If R² ≤ 0.99, repeat the entire procedure until R² exceeds 0.99.
  - Derive the linear equation of the usable line of best fit; this equation will be used to determine the glucose concentration of solutions with unknown concentrations.

Apparatus and Materials

Chemicals	Apparatus
Substrate solution	96-well microplate
Supernatant after cell lysis (protein samples)	8-strip PCR tubes
DNS reagent	Heated shaker

Procedure:
1. Prepare five strips of 8-strip PCR tubes, each corresponding to a different reaction time (0, 10, 20, 30, and 40 min). Dispense the appropriate volume of substrate into each tube. Place the tubes at 4 °C for about 10 min to pre-cool the substrate.

2. Prepare an ice box. Retrieve both the supernatant and the pre-cooled substrate from the fridge.

3. On ice, quickly add the designated volume of supernatant (protein sample) into each PCR tube. The total volume in each tube should be adjusted to 75 µL.

4. Immediately after adding the protein, add 75 µL of DNS reagent to the “0 min” tubes. Place the remaining tubes (with rack) into a pre-heated shaker at 37 °C and start timing.

5. At each time point (10, 20, 30, and 40 min), remove the corresponding PCR strip and promptly add 75 µL of DNS reagent.

6. Vortex all tubes for 2 min and centrifuge for 5 min.

7. Incubate the tubes at 100 °C in a PCR thermalcycler for 10 min to ensure complete color development.

8. Centrifuge again for 5 min to pellet any insoluble substrate.

9. Transfer 200 µL of the supernatant from each tube into a 96-well microplate and measure absorbance at 540 nm.

Apparatus and Materials

Chemicals	Apparatus
G250 solution	Vortex Mixer
BSA solution	Centrifuge
ddH2O	Spectrohotometer

Procedure:
1. Prepare BSA (a protein) solution of concentration of 1 mg/ml from any higher concentration solution.

2. Prepare wanted concentration using the table below.

3. Vortex is needed every time adding new solution.

Concentration(mg/mL)	0.00	0.02	0.04	0.06	0.08	0.10
BSA(1mg/mL) (ul)	0	1	2	3	4	5
ddH2O(ul)	50	49	48	47	46	45
Total volume	50	50	50	50	50	50

<<<<<<< HEAD
4. Mix 40µL of the sample and 200µL of G250, vortex. Transfer 200µL of the final mixture to corresponding wells of 96-well plate for measurement, with the wavelength of 595nm.

5. create standardized curve, setting y axis as the concentration and x axis as the optical density.

=======
4. Mix 40µL of the sample and 200µL of G250, vortex. Transfer 200µL of the final mixture to corresponding wells of 96-well plate for measurement, with the wavelength of 595nm.

5. Create standardized curve, setting y axis as the concentration and x axis as the optical density.

>>>>>>> bbc171f876e9a44a1e45660d5a659a79f956ad4c

Apparatus and Materials

Chemicals	Apparatus
Coomassie Brilliant Blue G-250	Pipette
Protein eluate	Vortex mixer
ddH2O	96-well plate
	Centrifuge tube (1.5 mL)
	Microplate reader

Procedure:
1. Combine aliquots from each eluate in proportion to their original volumes to generate a representative pooled protein sample in 1.5 mL centrifuge tube.

2. Mix 40 μL of the sample and 200 μL Coomassie Brilliant Blue G-250 in a new 1.5 mL centrifuge tube using vortex mixer; make sure precipitation is dissolved completely.

3. Transfer 200 μL mixture into 96-well plate; obtain its OD595 using a microplate reader.

4. Calculate the protein concentration using the standard curve.

5. If the concentration exceeds the standard curve range, dilute the sample from step 1 with ddH2O and redo step 2-4.

Apparatus and Materials

Chemicals	Apparatus
Coomassie Brilliant Blue G-250	Desktop Centrifuge
2% Chitin Solution	Vortex mixer
ddH2O	96-well plate
	Dry bath incubator
	Microplate reader

Procedure:
1. Add the following to a 2mL EP tube.

Reagent	Volume/μl
Supernatant	40
G250	200

2. Using a micropipette, suck out 200 μl into a 96-well plate to measure absorbance, wavelength should be 595nm.

3. If absorbance falls outside measurable range, dilute supernatant solution by twofold and re-measure, until absorbance falls within measurable range.



4. Vortex the chitin solution to ensure its mixed thoroughly.

5. Mix the following to a 2mL EP tube.

Reagent	Volume/μl
2% chitin solution	500
Supernatant	500

6. Dilute the solution by adding water, dilution factor should be half of the control.

7. Place the EP tube in a dry bath incubator set at 37 °C for 1 hour

8. Centrifuge the EP tube at 12000RPM for 10 minutes

9. Add the following to another 2mL EP tube

Reagent	Volume/μl
Supernatant and chitin solution	40
G250	200

10. Using a micropipette, suck out 200 μl into a 96-well plate to measure absorbance, wavelength should be 595nm.

11. Calculate difference between absorbance readings, and convert to standard curve.