Experiments | Shanghai-HS

Plasmid extraction (DiaSpin Plasmid Mini-Preps Kit)

Reagents	Amount
Buffer S	500μL
Buffer SP1	250μL
Buffer SP2	250μL
Buffer SP3	350μL
Wash solution	500μL
ddH₂O	50-100μL
Bacterial fluid	1.5-5mL

Apparatus	Amount
Spin column	1
Collection tube	1
Eppendorf tube (1.5mL)	2
Centrifuge	1
Vortex machine	1
Pipette (range: 1~5mL)	1
Pipette (range: 100~1000μL)	1

1. Insert one spin column into the collection tube

2. Add 500μL Buffer S into the column

3. Centrifuge the tube at 12000×g for 1 minute and discard the waste liquid

4. Put 1.5-5mL overnight cultured (37℃ 12 - 16h) bacteria into an Eppendorf tube

5. Centrifuge the tube at 8000×g for 2 minutes and discard the supernatant

6. Add 250μL Buffer SP1 to the bacteria precipitate

7. Blowing and vortex until bacteria are suspended

8. Add 250μL Buffer SP2 and immediately gently invert the tube 5-10 times

9. Rest the tube at room temperature for 2-4 minutes

10. Add 350μL Buffer SP3 and immediately gently invert the tube 5-10 times

11. Centrifuge the tube at 12000×g for 5-10 minutes

12. Put the supernatant into the spin column

13. Centrifuge at 9000×g for 30 seconds and discard the waste liquid

14. Add 500μL wash solution into the column

15. Centrifuge the tube at 9000×g for 30 seconds and discard the waste liquid

16. Repeat the steps 14&15.

17. Centrifuge the tube at 9000×g for 1 minute

18. Place the spin column into a new Eppendorf tube

19. Add 50-100 μL preheated ddH₂O (56℃) on the middle of the absorption membrane

20. Rest the tube at room temperature for 1-2 minutes

21. Centrifuge the tube at 9000×g for 1 minute

Restriction enzyme single digestion

Reagents	Amount
10×H Buffer	5μL
Nde1 enzyme	1μL
pBridge-BD-CRY2 plasmid	800ng
dH₂O	Up to 50μL

Apparatus	Amount
Eppendorf tube (1.5mL)	1
Water bath kettle	1
Small centrifuge	1
Vortex machine	1
Pipette (range: 1~10μL)	1
Pipette (range: 10~100μL)	1

1. Add 5μL 10×H buffer, 1μL Nde1 enzyme, 800ng pBridge-BD-CRY2 plasmid, up to 50μL dH₂O into the Eppendorf tube on ice

2. Vortex and centrifuge the tube

3. Place the tube into a water bath kettle at 37℃ for 15 minutes

Restriction enzyme double digestion

Materials

Reagents	Amount
10×H Buffer	5μL
10× rCutsmart Buffer	5μL
pBridge-BD-CRY2 plasmid	1μg
Nde1 enzyme	1μL
Bstb1 enzyme	1μL
dH₂O	Up to 50μL

Apparatus	Amount
Eppendorf tube (1.5mL)	1
Water bath kettle	1
Small centrifuge	1
Vortex machine	1
Pipette (range: 1~10μL)	1
Pipette (range: 10~100μL)	1

Protocol

1. Add 5μL 10×H buffer, 5μL 10× rCutsmart buffer, 1μg pBridge-BD-CRY2 plasmid, 1μL Nde1 enzyme, 1μL Bstb1 enzyme, up to 50μL dH₂O into the Eppendorf tube on ice

2. Vortex and centrifuge the tube

3. Place the tube into a water bath kettle at 37℃ for 2 hours

Touchdown PCR

Materials

Reagents	Amount
2× phata mix	25μL
Template (TRP1 promoter, UVR8 or BIC2 CDS)	1μL
Forward primer (TRP1-F, UVR8-F or BIC2-F, correspond to the template)	1μL
Reverse primer (TRP1-R, UVR8-R or BIC2-R, correspond to the template)	1μL
dH₂O	Up to 50μL

Apparatus	Amount
Eppendorf tube (1.5mL)	1
PCR tube	1
Small centrifuge	1
Vortex machine	1
PCR thermal cycler	1
Pipette (range: 0.5-2.5μL)	1
Pipette (range:10-100μL)	1

Protocol

1. Prepare 3 PCR tubes. Add 25μL phata mix, 1μL forward primer and 1μL reverse primer into each PCR tube.

2. Add template into each PCR tube.

3. Add water into each PCR tube up to 50μL

4. Vortex and centrifuge the tube.

5. Set up a reaction program.

Temperature	Time
94℃	5mins
94℃	30s
65℃	30s
72℃	1kb/min (depends on the template)
Repeat 10 cycles, decreasing by 1 ° C each cycle during the 65 ° C stage (65 to 55 ° C)
94℃	30s
55℃	30s
72℃	1kb/min (depends on the template)
Repeat 25 cycles
72℃	10mins

6. Start the PCR reaction.

Colony PCR

Materials

Reagents	Amount
Taq Mix	5μL
Forward primer (TRP-F)	0.5μL
Reverse primer (BIC2-R)	0.5μL
dH₂O	4μL
Template (incubated DH5α chemically competent cells with plasmids pBridge-BD-CRY2-UVR8-BIC2)	--
Liquid LB medium (amp+)	1mL

Apparatus	Amount
Eppendorf tube (1.5mL)	1
PCR tube	1
Small centrifuge	1
Vortex machine	1
PCR thermal cycler	1
Pipette (range: 0.5~2.5μL)	1
Pipette (range:1~10μL)	1
Pipette (range:1~5mL)	1

Protocol

1. Add 5μL Taq Mix, 0.5μL forward primer, 0.5μL reverse primer, 4μL dH₂O into a PCR tube.

2. Add 1mL ampicillin resistance liquid LB into a 1.5mL Eppendorf tube.

7. Pick a single clonal colony with a pipette (range: 0.5-2.5μL), and the tip must be blown out in the PCR tube for 3 times.

3. Vortex and centrifuge the tube.

4. Remove the tip from the PCR tube and put it into the1.5mL Eppendorf tube to culture bacteria.

5. Set up a reaction program.

Temperature	Time
95℃	10mins
94℃	30s
55℃	30s
72℃	1min55s
28 cycles
72℃	10mins
10℃	30min

6. Start the PCR reaction.

Homologous recombination

Materials

Reagents	Amount
2× Mix Buffer	10μL
pBridge-BD-CRY2 plasmid (8361bp)	167.22ng
TRP1 promoter fragment (134bp)	2.68ng
UVR8-N397 fragment (1213bp)	24.26ng
BIC2-CDS fragment (384bp)	7.68ng
dH₂O	Up to 20μL

Apparatus	Amount
Eppendorf tube (1.5mL)	1
Water bath kettle	1
Small centrifuge	1
Vortex machine	1
Pipette (range: 0.5~2.5μL)	1
Pipette (range:10~100μL)	1

Protocol

1. Add 10μL 2× Mix Buffer, 167.22ng pBridge-BD-CRY2 plasmid, 2.68ng TRP1 promoter fragment, 24.26ng UVR8-N397 fragment, 7.68ng BIC2-CDS fragment, up to 20μL dH₂O into an Eppendorf tube.

2. Vortex and centrifuge the tube.

3. Place the tube into a water bath kettle at 50℃ for 25min.

4. Place the tube on ice.

1% Agarose gel electrophoresis

Materials

Reagents	Amount
Agarose	1% (0.8μL)
1× TAE Buffer	80ml
Nucleic acid dye(1000x)	8μL
10× loading buffer	1× in template

Apparatus	Amount
Conical flask	1
Measuring cylinder	1
Weighing paper	1 piece
Electronic balance	1
Spatula	1
Electrophoresis chamber	1
Electrodes	1 pair
Gel tray	1
Comb	1
UV transilluminator	1
Pipette (range: 1~10μL)	1

Protocol

1. Place the weighing paper on the electronic balance, weigh 0.8g agarose and pour it into the conical flask.

2. Pour 80mL 1× TAE Buffer into the conical flask.

3. Heat the conical flask in the microwave oven for 1~2minutes until the agarose is well dissolved.

4. Add 8μL nucleic acid dye into the conical flask and shake it.

5. Pour the gel into the gel tray and insert the comb.

6. Rest the gel in room temperature for 30 minutes until it is solidified.

7. Pour about 1000mL 1× TAE Buffer into the electrophoresis chamber.

8. Carefully take out the gel and place it into the electrophoresis chamber.

9. Use the pipette (range: 1~10μL) to add 5μL Trans®️ Marker into the first hole of the gel.

10. Add loading buffer, 0.1× volume of the template, into the template using a pipette (range: 1~10μL), mix well by blowing and suction.

11. Carefully add 10 μL of each template into the following holes of the gel

12. Connect the electrodes, turn on the voltage to 130V and work for 10 minutes.

13. Place the gel into the UV transilluminator and turn on the UV light.

14. Comparing with the marker reference to determine the target section.

DNA gel extraction (Diamond kit)

Materials

Reagents	Amount
Agarose gel	--
Buffer B2	400μL
Wash Solution	500μL
ddH₂O	15μL

Apparatus	Amount
Scalpel	1
Eppendorf tube (1.5mL)	1
Spin column	1
Collection tube	1
Water bath kettle	1
Centrifuge	1
Pipette (range: 10~100μL)	1
Pipette (range: 100~1000μL)	1

Protocol

1. Use the scalpel to cut the target section of the gel and place the gel into the Eppendorf tube.

2. Add 400μL buffer B2, place the tube into a water bath kettle at 50℃ for 5-10 minutes to dissolve the gel.

3. Insert a spin column into a collection tube and transfer the dissolved gel into the spin column.

4. Centrifuge the tube at 8000×g for 30s and discard the waste liquid.

5. Add 500μL Wash Solution.

6. Centrifuge the tube at 9000×g for 30s and discard the waste liquid.

7. Repeat the steps 5&6.

8. Centrifuge the tube at 9000×g for 1 minute.

9. Place the spin column into a new Eppendorf tube (1.5mL).

10. Add 15μL ddH₂O into the spin column, rest at room temperature for 1 minute.

11. Centrifuge the tube at 9000×g for 1 minute, save the solution in the Eppendorf tube.

E.coli transformation

Materials

Reagents	Amount
pBridge-BD-CRY2-UVR8-BIC2 plasmid	5~10μL
DH5α chemically competent cell	100μL
Liquid LB medium (amp-)	900μL
Solid LB medium (amp+)	1 plate

Apparatus	Amount
Eppendorf tube (1.5mL)	1
Water bath kettle	1
Sterile spreading stick	1
Petri dish	1
Incubator	1
Shaker	1
Pipette (range: 1~10μL)	1
Pipette (range: 10~100μL)	1

Protocol

1. Thaw 100μL DH5α chemically competent cells in an Eppendorf tube on ice

2. Add 10μL pBridge-BD-CRY2-UVR8-BIC2 plasmid into the tube, flick the tube to mix

3. Rest the tube on ice for 30 minutes

4. Place the tube into a water bath kettle at 42℃ for 45 seconds and immediately place the tube on ice for 2~3 minutes

5. Add 900μL liquid LB medium (amp-) to the tube

6. Mix the solution uniformly, and incubate it in the shaker at 37℃, 200~250rpm for 1 hour

7. Preheat the solid LB medium (amp+) in a Petri dish with an incubator for 37℃

8. Centrifuge the tube at 2500×g for 5 minute, discard 900μL supernatant

9. Resuspend the bacteria and coat them uniformly on the solid LB medium (amp+) using a sterile spreading stick.

10. Turn the Petri dish upside down and incubate the cells at 37℃ for 12-16 hours.

S. cerevisiae transformation

Materials

Reagents	Amount
pBridge-AD-CIB1 plasmid	2μg
pBridge-BD-CRY2 plasmid	2μg
AH109 chemically competent cell	100μL
Carrier DNA (Salmon sperm DNA)	100μL
PEG/LiAc	500μL
Solid LB medium (-Trp/-Leu)	900μL
ddH₂O	50μL

Apparatus	Amount
Eppendorf tube (1.5mL)	1
Water bath kettle	1
Sterile spreading stick	1
Petri dish	1
Incubator	1
Pipette (range: 1~10μL)	1
Pipette (range: 10~100μL)	1

Protocol

1. Place the carrier DNA into a water bath kettle at 95℃ for 3 minutes and immediately place the tube on ice

2. Thaw 100μL AH109 chemically competent cells in an Eppendorf tube on ice

3. Add 2~5μg pBridge-AD-CIB1 plasmid, 2~5μg pBridge-BD-CRY2 plasmid, 10μL carrier DNA, 500μL PEG/LiAc into the tube, mix well by blowing and suction

4. Place the tube into a water bath kettle at 30℃ for 30 minutes, turn the tube upside down 6~8 times when 15 minutes has passed during the water bath

5. Place the tube into a water bath kettle at 42℃ for 15 minutes, turn the tube upside down 6~8 times when 7.5 minutes has passed during the water bath

6. Centrifuge the tube at 2500×g for 40 seconds, discard supernatant

7. Add 400μL ddH₂O and resuspend

8. Centrifuge the tube at 2500×g for 30 seconds, discard supernatant

9. Add 400μL ddH₂O and resuspend

10. Coat the mixture uniformly on the solid LB medium (-Trp/-Leu) using a sterile spreading stick

11. Turn the Petri dish upside down and incubate the cells at 29℃ for 48~96 hours until the diameter of single colony reaches 1mm

β-galactosidase activity assay

Materials

Reagents	Amount
YPD medium	8mL
Liquid LB medium (-Trp/-Leu)	5mL
Positive sample (incubated AH109 chemically competent cells with plasmids pBridge-AD-CIB1 pBridge-BD-CRY2)	2mL

Apparatus	Amount
Eppendorf tube (1.5mL)	1
Water bath kettle	1
Sterile spreading stick	1
Petri dish	1
Incubator	1
Pipette (range: 1~10μL)	1
Pipette (range: 10~100μL)	1

Protocol

1. The positive colonies of bacteria grown on the -Trp/-Leu solid SD medium were cultured in 5ml the -Trp/-Leu liquid SD medium (Amp) at 30℃, 250rpm, overnight (wrapped in tinfoil). During the period, use the bule light treatment to activate β-galactosidase expression.

2. Light treatment: Vortex the overnight culture tube for 0.5 - 1 min to disperse cell clumps. Transfer 2 ml of the overnight culture to 8 ml of YPD medium Immediately. Incubate fresh culture at 30°C for 3 - 5 h with shaking (230 -250 rpm) until the cells are in mid-log phase (OD600 of 1 ml = 0.5 - 0.8). Record the exact OD600 when you harvest the cells. During the period, use the bule light and UVB treatment to close β-galactosidase expression.

3. Harvest the cell by centrifuge at 14000 rpm(16000xg) for 30 seconds and discard the supernatant carefully. According to the number of bacteria or cells (10⁴): the extraction liquid volume (mL) is 500~ 1000:1 ratio (it is recommended that 5 million bacteria or cells be added to 1mL of the extraction solution), and the bacteria or cells are broken by ultrasound (ice bath, power 20% or 200W, ultrasonic 3s, 10s interval, repeat 30 times); 15000g centrifuge at 4℃ for 10min, take supernatant and put it on ice to be measured.

4. β-galactosidase activity was detected, and cells were collected: centrifuge at 700g for 5min, collect yeast cells into a centrifuge tube, and discard the supernatant; Add 1ml of the extract. Ultrasonic crushing of bacteria or cells (ice bath, power 20% or 200W, ultrasonic 3s, 10s interval, repeat 30 times); 15000g centrifuge at 4℃ for 10min, take supernatant and put it on ice to be measured.

5. Treatment of standard liquid: Dilute the standard liquid with distilled water to 200, 100, 50, 25, 12.5, 6.25, 0 nmol/ml.

6. Add each reagent in turn according to the protocol of kit.

Reagents	Test tube	Control tube	Standard tube
Reagent 1	200	-	-
ddH₂O	-	200	-
Reagent 2	250	250	-
Sample supernatant	50	50	-
Mix quickly and place in a water bath at 37℃ for 30 minutes
Standard sample	-	-	500
Reagent 3	1000	1000	1000
Thoroughly mixed, the absorption value A was measured at 400nm, and ΔA was A value of test tube minus A value of control tube. Each test tube shall be provided with a control tube.

7. Develop a standard curve. Determine the β-GAL activity of sample.