PROTOCOLS

Wet lab experiments

Kit-free plasmid miniprep

Day1
Prepare the following solutions:
Solution I - Resuspension Buffer
1. 25 mM Tris-HCl (pH 8)
2. 50 mM glucose
3. 10 mM EDTA
Store Solution I at 4°C
Solution II - Denaturing Solution
4. 0.2 N NaOH
5. 1.0% SDS
Store Solution II at room temperature
Solution III - Renaturing Solution (Potassium Acetate)
6. 120 ml 5M Potassium acetate
7. 23 ml glacial acetic acid
8. 57 ml of dH₂O
Store Solution III at 4°C

1. Grow 4 ml overnight cultures from single colonies of bacteria containing plasmid.
2. Add 1.0 ml of the stock culture to a 1.5 ml microfuge tube.
3. Centrifuge in microfuge tube at 12,000 g for 1 min.
4. Pour off the supernatant.
5. Repeat step2~step4 four times.
6. Resuspend the pellet in 100 μl of cold Solution I.
7. Vortex the solution for 2 mins or until all bacteria are fully resuspended.
8. Add 200 μl of Solution II and invert the tube carefully 5 times to mix the contents. The contents will become clear and thicker as the proteins and DNA are denatured.
9. Incubate solution on ice for 5 mins.
10. Add 150 μl of cold Solution III to each tube.
11. Mix by inverting several times.
12. Incubate tube on ice for 5 mins.
13. Centrifuge the tube for 5 mins at 12,000 g.
14. Collect the supernatant into a new tube by pipetting.
15. To your DNA solution ( 350 μl) , add 2 volumes 95% ethanol( 700 μl) and 1/10 volume of 3 M pH 4.8 Na-acetate(35 μl).
16. Invert the microfuge tube to mix.
17. Place the tube at -20°C overnight.

Day 2
18. Centrifuge solution at 12,000 rpm for 20 mins at 4°C.
19. Pour out the supernatant.
20. Open and invert the tubes on a paper towel to drain them out.
21. Wash pellet by adding 500 µl cold 70% ethanol.
22. Centrifuge solution 12,000 rpm for 5 mins at room temp.
23. Pour out the supernatant.
24. Dry with vacuum or by inverting over paper towel for 5-20 mins.
25. Resuspend dry DNA with TE (10 mM Tris-HCl pH 8, 0.1 mM EDTA).
26. Store DNA at -20°C.

Pichia Lithium Chloride Transformation

Preparation of Cells (Pichia pastoris)
1. Grow a 50 ml culture of Pichia pastoris in YPD at 30°C with shaking to an OD₆₀₀ of 0.8 to 1.0 (approximately 108 cells/ml).
2. Harvest the cells, distribution 25ml into two 50ml centrifuge tube and Centrifuge at 1500 g for 10 mins at room temperature.
3. Wash pellet with 25 ml of sterile water, and Centrifuge at 1500 g for 10 mins at room temperature.
4. Decant the water and resuspend the cells in 1 ml of 100 mM LiCl.
5. Transfer the cell suspension to a 1.5 ml microcentrifuge tube.
6. Pellet the cells at 12000g for 1 min and remove the LiCl with pipette.
7. Resuspend the cells in 400 μl of 100 mM LiCl.
8. Dispense 50 μl of the cell suspension into a 1.5 ml microcentrifuge tube for each transformation and use immediately.

Transformation (YPD)
1. Centrifuge the LiCl-cell solution from Step 7, above, and remove the LiCl with a pipette.
2. For each transformation sample, add the following reagents IN THE ORDER GIVEN to the cells.
a. 300 μl 40% PEG
b. 36 μl 1 M LiCl
c. 25 μl 2 mg/ml single-stranded DNA (We use RNA remain in DNA sample replace it)
d. Linear plasmid DNA in 50 μl sterile water
4. Vortex each tube vigorously until the cell pellet is completely mixed.
5. Incubate the tube at 30°C for 30 minutes without shaking.
6. Heat shock in a water bath at 42°C for 25 minutes.
7. Centrifuge the tubes at 8000 rpm and remove the transformation solution with a pipette.
8. Resuspend the pellet in 1 ml of YPD and incubate at 30°C with shaking.
9. After 1 hour and 4 hours, plate 100 μl on YPD plates containing 50 μg/ml Zeocin™.
10.Incubate the plates for 2–3 days at 30°C.

Transformation (homemade E. coli DH5α)

1.Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins)
2.Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator.
3.Mix 5 μl of DNA into 50 μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times.
4.Incubate the competent cell/DNA mixture on ice for 30 mins
5.Heat shock each transformation tube into a 42°C water bath for 90 secs.
6.Put the tubes back on ice for 5 mins.
7.Add 50 μl LB to the bacteria and grow in 37°C shaking incubator for 180 mins
8.Spin down
9.Plate all of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic (Chloramphenicol).
10.Incubate plates at 37°C overnight.

Creating the gel

1.Use 1.0 % agarose from common stock to pour gels. If unavailable or another percentage is desired use 1.0 g Agarose per 100 ml 1x TAE for 1.0 %.
2.Mix and heat in microwave till all agarose powder is dissolved.
3.Pour liquid gel into mold and wait ~15 mins for it to solidify, longer if using a lower agarose concentration.

Gel Extraction

1. After running the DNA gel in TAE buffer, cut out the desired DNA band with a clean scalpel. Weight the gel slice in a microcentrifuge tube.
2. Add equal volum of Binding Buffer (100mg gel slice add 100 µL )
3.Incubate at 60℃ for 10 mins and vortex the mixture , until the gel slice is completely dissolved.
4. Insert the Spin column into a Collection tube, transfer the solution into the spin column and spin at 12000rcf/ 1 min, and discard the flowthrough liquid in the collection tube.
5. Add 600 µl of Washing Buffer and centrifuge at 12000rcf/ 1 min. Repeat this wash procedure for two times.
6. Discard the flowthrough liquid and centrifuge for 3 mins at full speed to remove residual trace of ethanol.
7.Transfer the spin column into a new microcentrifuge tube and add 27µl of Elution Buffer wait for 10 min.
8. Centrifuge at 12000rcf/ 1 min to elute the DNA. Store the DNA in the microcentrifuge tube at -20℃.

DNA Clean-Up

1. Add equal volume(35µl) of Binding Buffer to solution.and vortex briefly to mix.
2. Insert the Spin column into a Collection tube, transfer the solution into the spin column and spin at full speed (12,000 x g) for 1 min, and discard the flow through liquid in the collection tube.
3. Add 600 µl of Washing Buffer and centrifuge at full speed for 1 min. Repeat this wash procedure for one more time (3min).
4. Discard the flow through liquid and centrifuge for 5 mins at full speed to remove residual trace of ethanol.
5. Transfer the spin column into a new microcentrifuge tube and add 35µl of Elution Buffer and wait for 10 mins.
6. Centrifuge at full speed for 1 min to elute the DNA.

YPD media

1.To an autoclavable flask, add:

2.Autoclave the mixture.
3.Prepare as appropriate:

For liquid media

a. Add 50 ml of sterile 40% (w/v) glucose. Mix.
b. Allow to cool before use

For agar plates

a. While stirring the autoclaved mixture on a magnetic stir plate, add 50 ml of sterile 40% glucose per liter of media.
b. While still warm, pour media into 9-cm diameter plastic Petri dishes.
c. Allow agar mixture to cool and solidify.

LB media

1. Add ~80% of the final volume of dH₂O to a sterile media bottle.
2. Add 1% NaCl, 1% Tryptone and 0.5% yeast extract.
3. Seal the cap of the media bottle and shake until components are completely dissolved.
4. Adjust the final volume using dH₂O.
5. SOLID MEDIUM ONLY Add the appropriate amount of agar to the solution.
6. Apply autoclave tape to the bottle and autoclave.

Standard Minimal Media (SD)

- autoclave 20 mins

- autoclave 20 mins, cool to<70℃, then add:

- mix well, store at 4℃ until use

Ligation protocol

1. Add appropriate amount of deionized H₂O to sterile 0.6 m tube
2. Add 1 μl ligation buffer to the tube.
Vortex buffer before pipetting to ensure that it is well-mixed.
Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.
3. Add appropriate amount of insert to the tube.
4. Add appropriate amount of vector to the tube.
5. Add 0.5 μl ligase.
Vortex ligase before pipetting to ensure that it is well-mixed.
Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 0.5 μl, just touch your tip to the surface of the liquid when pipetting.
6. Let the 10 μl solution sit at 22.5°C for 30 mins
7. Denature the ligase at 65°C for 10mins
8. Dialyze for 20 mins if electroporating
9. Use disks shiny side up
10. Store at -20°C

Run electrophoresis gel

1. Make a suitable agarose gel (depend on your DNA size), X (g) agarose + Y (ml) 0.5X TAE, X/ Y = 1.2%~1.5% (<0.5k), heating until all agarose melted.
2. Cool down the agarose gel (use the back of your hand to check it, as long as it does not burn your hand, it is suitable temperature), add EtBr (final, 5 μl/ 100 ml) into agarose gel and mix well.
3. Put agarose gel into mold and wait until it become solidification.
4. Load samples into each well, the recipe is show below.
5. Run gel in electrophoresis tank under 100 V.

For agar plates

a. While stirring the autoclaved mixture on a magnetic stir plate, add 50 ml of sterile 40% glucose per liter of media.
b. While still warm, pour media into 9-cm diameter plastic Petri dishes.
c. Allow agar mixture to cool and solidify.

PCR

Strain Preservation and Revival Yeast strains

Strain Preservation Yeast

1.To prepare glycerol stocks, make a sterile solution of 30% glycerol (w/v).
2.Pipet 1.0 ml of the solution into sterile 4-ml screw-cap vials.
3.Add 1.0 ml of a late log or early stationary phase culture, mix, freeze on dry ice,and store at –70°C.
4.Revive the strain by scraping some cells off the frozen surface and streak onto plates. It is not necessary to thaw the entire vial.

Revival Yeast strains

1. Dip the paper into a yeast culture or press onto a yeast colony using sterile forceps.
2. Then wrap the filter paper in sterile aluminum foil and mail.
3. The strain is revived by placing the paper onto the surface of an agar plate and incubating the plate at 30°C for several days.

Western blot protocol

Sample preparation

Pellet:
1. Centrifuge 1ml YPD at 5000rpm for 10 mins.
2. Wash the cell pellet by gently resuspending (with pipette) in 1.2 M Sorbitol solution, before lyticase treatment and spinning at 5000rpm for 10 mins.
3. Dilute 10 times stock solution of lyticase in Z buffer solution. 1ml of lyticase in Z buffer per 1g of cell pellet is added in each tube for 30 mins at room temperature. Do not use vortex at this step. The temperature is very important for an optimal lysis so that an incubator should be used if the room temperature was higher than 22°C.
4. Collect the spheroplasts by centrifugation at 1,000 x g, 5 mins 4℃.
5. Add 50μl ddH₂O into pellet, resuspend, and add 50μl 2x Laemmli Sample Buffer, boil 100℃/ 5mins, store at -20℃.
6. Boil 100℃/ 5mins before loading.

Z buffer(1L):
16.1 g Na2HPO4 7 H₂O (60 mM final)
5.5 g NaH2PO4 H₂O (40 mM final)
0.75 g KCl (10 mM final)
0.246 g MgSO4 7H₂O (1 mM final)
2.7 ml β-mercaptoethanol (50 mM final) Adjust to pH 7.0. Do not autoclave

Soup:
1. Insert the Amicon® Ultra-0.5 device into one of the provided microcentrifuge tubes.
2. Add up to 500 µL of sample to the Amicon® Ultra filter device and cap it.
3. Place capped filter device into the centrifuge rotor, aligning the cap strap toward the center of the rotor; counterbalance with a similar device.
4. Spin the device at 14,000 × g for approximately 10–30 minutes depending on the NMWL of the device used. Refer to Figure 1 and Table 2 for typical spin times.
5. Remove the assembled device from the centrifuge and separate the Amicon® Ultra filter device from the microcentrifuge tube.
6. To recover the concentrated solute, place the Amicon® Ultra filter device upside down in a clean microcentrifuge tube. Place in centrifuge, aligning open cap towards the center of the rotor;counterbalance with a similar device. Spin for 2 minutes at 1,000 × g to transfer the concentrated sample from the device to the tube. The ultrafiltrate can be stored in the centrifuge tube.
7. There are about 200μl remain. The samples are concentrate about 5 times.
8. Add 5μl 2x Laemmli Sample Buffer into 5μl sample, Boil 100℃/ 5mins before loading.

SDS-PAGE

We use 10% SDS-PAGE, 10μl sample per well, run at 60V until the sample over the Stacking gel, and run at 90V in Separating gel until the blue front is at thebottom of the gel.

Transfer

1. Activate PVDF with methanol for 1 min and rinse with transfer buffer before preparing the stack.
2. Prepare the stack form negative electrode to positive electrode as follow: sponge> filter paper> gel> membrane> filter paper> sponge.
3. Transfer at 400mA at 90 mins.

Blocking

1. Prepare 5% milk in PBST, blocking at room temperature 1 hr.
2. Wash with wash buffer (0.5% milk in PBST) at room temperature 10 mins 3 times.

Antibody staining

1. Dilute primary antibody in wash buffer.
(mouse anti-HA: 1/2000 mouse anti-FLAG: 1/1000 mouse anti-V5: 1/5000)
2. Incubate at 4℃ overnight.
3. Wash with wash buffer at room temperature 10 mins 3 times.
4. Dilute secondary antibody in wash buffer. (anti-mouse HRP: 1/10000)
5. Incubate at room temperature 1 hr.
6. Wash with wash buffer at room temperature 10 mins 3 times.

FPLC protocol

Disrupt cell wall of yeast (French press method)
1. Prechill the liquid culture at 4°C, centrifuge the culture at 4°C, 4000 g, for 10 mins
2. Resuspend the cells in chilled His binding buffer. (Ratios of cell wet weight to buffer volume of 1:1)
3. Apply the sample to the French pressure cell and bring the cell under the desired pressure (7000 to 10,000 psi)
4. While maintaining the pressure, adjust the outlet flow rate to about one drop every second. Collect the cell lysate in a flask that is kept on ice
5. Repeat steps 3 and 4 twice.
6. Remove cell debris by ultracentrifugation at 4°C for 20 mins at 12,000 g.

Preparation of Ni-Sepharose Beads
1. Prepare 25 ml of beads and wash the beads with mQH₂O three times, NiSO4 once, mQH₂O three times, acetic acid once, and mQH₂O three times.
2. Wash with His binding Buffer one time.
3. Mix the beads with the supernatant.

Binding (Batch Binding Method)
4. Save 100 μl of lysate. Add clarified lysate (if frozen, check for ppt material. If there is any clumpy or ppt material or if the lysate is cloudy, centrifuge and keep the supernatant) to the washed beads.
5. Combine the washed beads and lysate onto a drained and capped column or a 50 ml falcon tube for smaller volumes.
6. Tightly cover with parafilm and incubate rocker for 30 mins at room temp.
7. Replace the column on the stand and allow most of the beads settle then open column.
8. Add frit back to column, re-wash the flow thru.

After binding
9. Wash beads ~ 16 ml of Ni-Column His Binding Buffer. Save as flow through wash in one fraction.
10. Wash the column with ~500 ml of His Wash Buffer. *This will remove some of the weakly binding protein.
11. Continue with the His wash buffer until no detectible protein is found in eluate.
12. Elute the protein with 20 x 15 ml of His Elution Buffer.
13. Check each fraction for total protein and determine purity by SDS-PAGE – coomasie stain

His-Binding Buffer:
50 mM Tris-Cl (pH8.0)
100 mM NaCl
5 mM Imidazole
include 1 mM PMSF made fresh

His-Wash Buffer:
50 mM Tris-Cl (pH8.0)
300 mM NaCl
10-20 mM Imidazole
Include 1mM PMSF made fresh

His-Elution Buffer:
50 mM Tris-Cl (pH8.0)
50 mM NaCl
300 mM Imidazole
include 1mM PMSF made fresh

Reaction experiments

Prepare the following solutions

Solution I – Peroxidase buffer(1)
1. 50mM hydrogen peroxide (store at 4°C)

　Solution II - Peroxidase buffer(2) [pH5]
1. 250mM sodium acetate
2. 12.5mM calcium chloride
3. 156.25mM sodium chloride
4. a few hydrochloric acid

　Solution III -Laccase buffer
1. 250mM sodium acetate
2. 12.5mM cupric chloride
3. 156.25mM sodium chloride

　Solution IV -Coniferyl alcohol buffer
1. 340 mM Coniferyl alcohol in 95% ethanol

Protocol

1. Take some liquid of yeast to centrifuge at 4500 × g at 4°C.
2. Put supernatant in 10kDa microcentrifuge tube, and centrifuge at 4250 × g for an hour at 4°C.
3. Take
1ml Solution I,
1ml Solution II,
1ml Solution III,
0.5ml peroxidase x16 concentrated solution (higher than 10kDa),
0.5ml peroxidaes x18 concentrated solution (higher than 10kDa),
0.5ml laccase concentrated solution (higher than 10kDa)
And 0.5ml Solution IV.
Put the buffer into glass tube and cover it, setting and shaking the tube in the 25°C incubator at 150 rpm for 6 hours.
5. Take the solution; counterbalance with similar device.
6. Spin the device at 4500 × g for 30 minutes.
7. Pour off the supernatant.
8. Put the pellet in the -80°C refrigerator.
9. Take the frozen solution into the lyophilization machine for a day to remove water.
10. Finally, getting the product which is white powder.

Ultraviolet-visible spectroscopy, UV-Vis

1. Take 200μl solution.
2. Counterbalance with similar device at 4500 × g for 30minutes.
3. Pour off the supernatant.
4. Take the solution at 0, 1 ,2, 3, 4 hours; repeat the steps 1, 2, 3.
5. Put some sodium hydroxide to dissolve the pellet.
6. Use the UV-Vis machine.

Team:CCU Taiwan/Protocols

PROTOCOLS