Team:BSC United/Experiments

PROJECT

EXPERIMENTS

Gene Fragment PCR Amplification

The PCR is an effective procedure for generating large quantities of specific DNA sequence in vitro. This amplification is achieved by a three-step cycling process.

Reaction Mixture	20µL
2x Mix (Taq DNA polymerase (0.05 U/µL), reaction buffer, 4 mM MgCl2, and 0.4 mM of each dNTP)	10µL
ddH2O	7µL
Template DNA	2µL
Primer R	0.5µL
Primer F	0.5µL

1. Make the reaction mixture with the above recipe in 1.5 mL Eppendorf tubes
2. Use microcentrifuge to make the liquid in Eppendorf tubes homogenous
3. Put the Eppendorf tubes into the PCR machine and start the thermocycling process with the parameters below:
1. 94˚C for 3min
2. b.94˚C for 30s
3. 48˚C for 30s
4. 72˚C for 30s
5. Cycle for 30 times
6. 72˚C for 10min
7. 4˚C storage

Colony PCR

Reaction Mixture	20µL
2x Mix (Taq DNA polymerase (0.05 U/µL), reaction buffer, 4 mM MgCl2, and 0.4 mM of each dNTP)	10µL
ddH2O	7µL
DNA Dilution	2µL
Primer R	0.5µL
Primer F	0.5µL

1. Pick a single colony with pipette tip from the solid growth on the plate
2. Dilute them with 5µl ddH₂O in an Eppendorf tube
3. Make the master mix with 2µL of the diluted DNA
4. Put the whole mixture in Eppendorf tubes into the PCR machine and start the thermocycling process with the parameters below:
1. 94˚C for 3min
2. b.94˚C for 30s
3. 48˚C for 30s
4. 72˚C for 30s
5. Cycle for 30 times
6. 72˚C for 10min
7. 4˚C storage

Gel Preparation

Agarose	1.2g
0.5x TAE	100mL
Goldview I (Nuclear Staining Dye)	3µL

1. Weigh the agarose
2. Add TAE and agarose together
3. Heat the mixture in the microwave
4. Cool for 30min
5. Add Goldview I
6. Pour it into the mold
7. Cool for 30min

Gel Electrophoresis

Material: TAE buffer

1. Immerse the gel into the chamber
2. Pour the TAE buffer until it completely covers the gel
3. Add 2µL of DNA into each well
4. Put the marker into the leftmost lane
5. Connect the electrodes
6. Turn on the power supply to 90v for 30 minutes
7. Put the gel under UV light and observe

Gel Extraction

1. Cut the desired gel fragment and place it into a tube
2. Add equal weight of Binding Buffer as the gel (XP2)
3. Vortex
4. Place the tube into 55˚C water bath for 10 minutes
5. Vortex
6. Add 300µL of Binding Buffer into the tube and centrifuge it for 1 minute at 12000rpm
7. Discard the filtrate
8. Add 700µL of SPW Wash Buffer into the tube and centrifuge it for 1 minute at 12000rpm
9. Discard the filtrate
10. Repeat steps 9~10 once
11. Centrifuge for 3 minutes at 12000rpm
12. Use 25µL of ddH₂O to wash down the pellet
13. Let it stand for 2 minutes
14. Centrifuge it for 1 minute at 12000rpm

Restriction Digest

Reaction Mixture	20µL
10x Buffer	2µL
BamH I	1µL
EcoR I	1µL
ddH2O	~up to 20µL
DNA Fragments	1µg

1. Make the mixture in a 1.5 mL Eppendorf tube
2. Put it into the PCR machine for 15 minutes at 37˚C

Nanodrop UV Spectrophotometer

1. Use distilled water and filter paper to clean the nanodrop UV spectrophotometer
2. Calibrate it using distilled water
3. Use the wipes to dry it completely
4. Pipette 1µL of DNA sample onto the platform
5. Record the measurements

Ligation

Reaction Mixture	10µL
ddH2O	~up to 10µL
Insert DNA	50ng
10x Buffer	1µL
T4 Ligase	0.5µL
Vector	10ng

1. Make the mixture in a 1.5 mL Eppendorf tube
2. Put it into the PCR at 16˚C for 3 hours

Revival of Bacteria

GM I	10mL
ddH2O	8.604mL
10% conc. Yeast Extract	1mL
10x C-Salts	956µL
20% conc. Glucose Solution	250µL
10mg/mL Amino Acid Solution	50µL
5% conc. Casein Hydrolysate	40µL

1. inoculate bacteria into the culture dishes that contain antibiotic and grow overnight at 37 ˚C
2. Make GMI
3. Transfer the cells from the culture dishes onto the GMI
4. Incubate at 30˚C 150rpm overnight

Transformation of E. Coli

1. Mix 10µL of plasmid with 100µL of E. Coli in a 1.5mL Eppendorf tube
2. Ice bath it for 30 minutes
3. Put it into hot water bath for 45 seconds at 42˚C
4. Ice bath it for 2 minutes
5. Mix with 500µL LB medium
6. Shake the bacteria culture for 30 minutes at 37˚C with 200rpm
7. Spread the bacteria culture onto plates

Transformation of Bacillus subtilis

GM II	100 mL
ddH2O	83.95 mL
10× C-Salts	9.328mL
20% conc. Glucose Solution	2.5mL
1M MgCl2	2mL
1M CaCl2	2mL
10 mg/mL Amino Acid Solution	0.1mL
5% conc. Casein Hydrolysate	0.08mL
10% conc. Yeast Extract	0.04mL

1. Make GMII
2. Add 5 mL of B. subtilis into GM II
3. Shake the mixture for 1.5 hours at 120 rpm
4. Centrifuge the mixture for 10 minutes at 500g
5. Discard the supernatant
6. Resuspend pellet in 5 mL fresh medium
7. Discard the other supernatant
8. Add the plasmid into the bacterial culture medium
9. Shake the mixture for 1 hour at 150 rpm
10. Centrifuge the mixture for 5 minutes at 5000 rpm
11. Resuspend the pellet in 100 µL fresh medium
12. Discard the other supernatant
13. Spread the growth medium onto plates and incubate overnight at 37 °C

Protein Gel Electrophoresis

	Running Gel	Stacking Gel
30% Acrylamide	5mL	0.33mL
Tris-HCl	2.5mL (1.5M, pH 8.8)	0.25mL (1M, pH 6.8)
ddH2O	2.3mL	1.4mL
10% SDS	0.1mL	0.02mL
10% APS	0.1mL	0.02mL
TEMED	0.004mL	0.002mL

1. Make the gel
2. Mix 4 µL of 5x SDS loading buffer and 16 µL culture medium
3. Water bath for 10 minutes at 100°C
4. Put the gel into the device
5. Load the samples into the gel
6. Turn on the power supply at 60V until the samples reach the running gel
7. Turn up the voltage to 120V for 90 minutes
8. Dye the gel with G-250 solution (100mg G-250, 50 ml 95% ethanol, 100 ml 85% phosphoric acid)
9. Shake and wash with destaining solution (50ml methanol, 75ml ethanoic acid, 875ml ddH₂O

Protein Extraction (lysozyme)

1. Centrifuge the bacteria in 40 ml of cell culture at 12000 rpm for 1 minute
2. Discard the supernatant
3. Add 10 ml ddH₂O
4. Distribute into 9 test-tubes
5. Centrifuge at 12000 rm for 1 minute
6. Discard the supernatant
7. Add 100 µL lysozyme
8. Water bath at 37 °C for 60 min

Protein Extraction (extraction kit)

Material: Invent Biotech Minute™ Total Protein Extraction Kit

1. Centrifuge cell culture
2. Add 1 ml water
3. Centrifuge at top speed in a microcentrifuge for 2 min
4. Remove supernatant completely
5. Resuspend the pellet in 50 µl buffer A
6. Weigh out 80-90 mg protein extraction powder and add to the bottom of the tube
7. Grind the tube repeatedly with pestle for about 2 min with twisting force
8. Add 50 µl buffer A and 100 µl buffer B to the same tube and continue to grind for about thirty seconds
9. Vortex vigorously for 10 seconds
10. Centrifuge the tube at top speed for 3-4 min
11. Proteins will be in the supernatant

Bradford Assay

Materials:

• Bradford solution

Bradford Solution	1000mL
Coomassie Brilliant Blue G-250	100mg
95% ethanol	50 mL
85% phosphoric acid	100 mL
ddH2O	~up to 1000 mL

• BSA standard solution 0.1 µg/µl
• 0.1% NaCl Solution

1. Prepare the following protein solution

Test-tube	0	1	2	3	4	5	6
BSA standard solution /mL	0.0	0.1	0.2	0.4	0.6	0.8	1.0
0.1% NaCl Solution	1	0.9	0.8	0.6	0.4	0.4	0.0
Bradford solution /mL	5	5	5	5	5	5	5

2. Use the microcentrifuge for the solution to be thoroughly mixed
3. Let them rest for 5 minutes
4. Measure absorbance at 595 nm
5. Prepare a calibration cure by plotting absorbance against protein amount in µg
6. Determine the amount of protein in our sample using the equation of the curve

Preparation of Competent Cells

1. Inoculate the yeast into YPD broth, and shake for overnight at 30˚C and 200rpm
2. Transfer 5 mL from the broth into 100 mL YPD and shake at 30˚C and 200rpm
3. Collect the yeast when the OD value lies between 1.2 and 2.0.
4. Collect yeast after 3000rpm for 5min
5. Centrifuge at 1600g for 5min at 4˚C
6. Discard the supernatant
7. Resuspend yeast with 10 mL 1mol/L D-Sorbitol suspension
8. Centrifuged at 1600g for 5 min at 4˚C
9. Discard the supernantent
10. Repeat step 7~9 five more times
11. Water bath the yeast in cool water

Electrotransformation of Yeast

1. Add 80 μl of competent yeast cells onto pre-chilled electroporation cuvette.
2. Mix 20 μl of concentrated linearized plasmid solution with the yeast cells
3. Place on ice for 5 min
4. Start the electroporation process
5. Immediately add 1 mL pre-cooled sterile 1 mol/L D-Sorbitol and rest for 60 minutes
6. Spread on MD plate
7. Place in 30℃ oven for 20-60 min
8. Incubate for 3-5 days

Colometric Assay

Material: 100 mL of the product of ASN hydrolysis

1. Add 3400μl deionized water to 100ml solution
2. Add 500μl Nessler's reagent
3. Carry out the nanodrop to find out the absorbance value

Ingredients of Medium

For Bacillus subtilis cultures:
LB Medium

LB	1L
Tryptone	10g
NaCl	10g
Yeast Extract	5g
Agar (for solid medium)	15g

For yeast culture:
YPG Medium

YPG	1L
Tryptone	20g
Yeast Extract	10g
Glycerol	20mL
Agar	20g

YPD Medium

YPD	1L
Tryptone	20g
Yeast Extract	10g
Glucose	20g
Agar	20g
ddH2O	~up to 1L

For yeast fermentation:
BMGY

BMGY	1L
Tryptone	20g
Yeast Extract	10g
1.34% YNB	100mL
2% glycerol	50mL
10 mmol/L potassium phosphate (pH 6.0)	100mL
Biotin 4×10-5%	2mL
ddH2O	~up to 1L

BMMY

BMMY	1L
Tryptone	20g
Yeast Extract	10g
1.34% YNB	100mL
0.5% methanol	100mL
10 mmol/L potassium phosphate (pH 6.0)	100mL
Biotin 4×10-5%	2mL

Ⓒ 2019 iGEM BSC_United