PCR of the target Gene

A. Materials

ddH2O	32 μL
dNTP	5 μL
MgSO4	4 μL
Forward primer	1 μL
Reverse primer	1 μL
DNA template	1 μL
KOD+ buffer	5 μL
KOD+	1 μL

B. Protocol

1. Prepare and mark PCR tubes and align tubes in order on tube rack.

2. Under normal circumstances, ddH2O should be added into tubes first, and enzymes should be added last. All other materials uncluding, but are not limited to dNTP, primers, buffers, and templates should be added in between.

3. PCR tubes should then be centrifuged briefly to ensure all liquids collect at the bottom of the tube.

4. Move PCR tubes to PCR machine to initiate the process.

C. Reacting conditions

98 °C	10 minutes	1 cycle
98 °C	15 seconds	26 cycles
55 °C	15 seconds
68 °C	1 minute

68 °C

10 minutes

1 cycle

4 °C

Infinite

N/A

Gel Electrophoresis of the Gene PCR Product A. Materials

Loading buffer & DNA dye

1 μL

PCR product

5 μL

TAE buffer

200 mL

Agarose

2 g

B.      Protocol

1.       Mix TAE buffer and agarose powder in a ratio of 100 mL TAE buffer : 1 g agarose powder.

2.       Heat the mixed solution in a microwave until agarose powder completely dissolves into TAE buffer.

3.       Pour the liquid into gel trays of suitable sizes. Insert a comb onto the tray to

4.       Mix 6x loading buffer premixed with DNA dye with PCR product in a ratio of 1 unit loading buffer : 5 units PCR product.

5.       Load proper ladder into first well of gel.

6.       Load samples into wells.

7.       Perform electrophoresis at 185 V for 10 minutes.

8.       Harvest gel, take image of gel under untraviolet light, and analyze results.

PCR Product Clean Up

A. Protocol (MN NucleoSpin Gel and PCR Clean-up Kit)

1. Combine obtained PCR products into one centrifuge tube.

2. Add buffer NTI to the product in a ratio of 2 μL buffer NTI : 1 μL PCR product to allow DNA to bind to the membrane.

3. Transfer the solution to a column placed within a collection tube. Centrifuge

at 12000 rpm for 30 seconds.

4. Transfer the liquid in the collection tube to the column. Centrifuge at 12000 rpm for 30 seconds.

5. Dispose of the liquid in the collection tube. Add 700 μL buffer NT3 to wash the membrane. Centrifuge at 12000 rpm for 30 seconds.

6. Repeat step 5.

7. Add 30 μL ddH2O preheated to 65 ˚C to the column. Centrifuge at 12000 rpm for 30 seconds.

B. Buffers

Buffer NTI: Adjust DNA binding conditions and allow DNA to bind to membrane.

Buffer NT3: Wash membrane.

Plasmid Extraction

A. Protocol (TIAN Prep Mini Plasmid Kit)

1. Harvest and transfer cultivated bacteria into marked 2 mL centrifuge tubes.

2. Centrifuge tubes at 12000 rpm for 30 seconds to deposit bacteria to the bottom of the tube. Dispose of the clear liquid on top.

3. Add 250 μL buffer P1 into the 2 mL centrifuge tubes to levitete the bacteria persent in the tubes.

4. Add 250 μL buffer P2 to the tubes to lyse bacterial cell wall. Mix the solution gently; do not vortex.

5. Add 350 μL buffer P3 to the tubes to neutralize buffer P2 and cause proteins in the solution to percipitate. Mix the solution gently; do not vortex.

6. Centrifuge the solution at 12000 rpm for 10 minutes to collect percitipation to the bottom of the tube.

7. Add 500 μL buffer BL to a column placed within a collection tube to wash the membrane. Centrifuge at 12000 rpm for 30 seconds.

8. Transfer the clear liquid obtained in step 6 to the column washed in step 7 and centrifuge the column at 12000 rpm for 30 seconds.

9. Dispose the clear liquid in the collection tube. Add 500 μL buffer PD to the column and centrifuge at 12000 rpm for 30 seconds.

10. Dispose of the liquid in the collection tube. Add 600 μL buffer PW to the column. Centrifuge at 12000 rpm for 30 seconds.

11. Repeat step 10.

12. Dispose of the clear liquid in the collection tube. Centrifuge at 12000 rpm for 1 minute.

13. Transfer the column to a new 2 mL centrifiuge tube. Dispose of the collection tube.

14. Add 40 μL ddH2O preheated to 65 °C to the column. Centrifuge at 12000 rpm for 30 seconds.

B. Buffers

Buffer P1: Levitate bacteria and lyse RNA. Buffer P2: Lyse bacteria cell wall.

Buffer P3: Neutralize P2 and cause proteins to precipitate. Buffer BL: Wash memnbrane pre-use.

Buffer PD: Wash proteins off the membrane. Buffer PW: Wash salts off the membrane.

Restriction Enzyme Digestion

A. Materials

ARSA/ARSR/ARSD
10x NEB buffer	5 μL
ARSA/D/R Plasmid (300 ng/μL)	10 μL
BsaI restriction enzyme	1 μL
ddH2O	34 μL
amilGFP
10x NEB buffer	5 μL
amilGFP (400 ng/μL)	10 μL
BsaI	1 μL

DpnI

1 μL

ddH2O

33 μL

B. Protocol

1. Mix the materials in a 1.5 mL centrifuge tube.

2. Place the centrifuge tube in 37 °C metal bath for at least 15 minutes.

Gel Electrophoresis of Restriction Enzyme Digestion Product

A. Materials

Loading buffer with DNA dye

10 μL

Digestion product

50 μL

B. Protocol

1. Mix 6x loading buffer premixed with DNA dye with PCR product in a ratio of 1 unit loading buffer : 5 units PCR product.

2. Load proper ladder into first well of gel.

3. Load samples into wells.

4. Perform electrophoresis at 185 V for 10 minutes.

5. Harvest gel, take image of gel under untraviolet light, and analyze results.

Agarose Gel DNA Extraction

A. Protocol (MN NucleoSpin Gel and PCR Clean-up Kit)

1. Collect gel with desired DNA fragments into 2 mL centrifuge tubes. Weigh the mass of gels on an electronic balance.

2. Add buffer NTI to the tube in a ratio of 2 μL buffer NTI : 1 mg agarose gel.

3. Heat the mixture at 50 ˚C in a metal bath until the gel dissolves completely.

4. Transfer the solution to a new column placed within a collection tube. Centrifuge at 12000 rpm for 30 seconds.

5. Transfer the liquid in the collection tube to the column. Centrifuge at 12000 rpm for 30 seconds.

6. Dispose of the clear liquid in the collection tube. Add 700 μL buffer NT3 to

the column. Centrifuge at 12000 rpm for 30 seconds.

7. Repeat step 6.

8. Dispose of the liquid in the collection tube. Centrifuge at 12000 rpm for 1 minute.

9. Add 30 μL ddH2O preheated to 65 ˚C to the column. Centrifuge at 12000 rpm for 30 seconds.

T4 DNA Ligase Ligation

A. Materials

ARSA/R/D plasmid (30 ng/μL)	1.5 μL
GFP (100 ng/μL)	4.5 μL
T4 DNA ligase	1 μL
T4 DNA ligase buffer	1 μL
ddH2O	2 μL

B. Protocol

1. Add ddH2O, T4 DNA ligase buffer, and T4 DNA ligase into a PCR tube with GFP gene and ARS plasmid in a ratio of 1 units ARS plasmid : 10 unit GFP gene.

2. Transfer the solution to PCR machine at 16˚C.

Transformation of DH5-α E. coli Using Heat Shock

A. Protocol

1. Mix the tube introduced DNA material with DH5-α competent cells. The volumn of DNA material must not exceed 10% of that of the competent cells.

2. Gently fick the tube 4-5 times to mix competent cells and DNA material. Do not vortex.

3. Place the tube on ice for 30 minutes.

4. Place the tube at exactly 42 ˚C for 90 seconds to perform heat shock.

5. Immediately place the tube back on ice after heat shock. Keep the tube on ice for 2-3 minutes.

6. Add 900 μL LB culture medium without KANA antibiotics to the tube. Incubate the transformed bacteria at 37 ˚C for 30 minutes.

7. Centrifuge the tube at 12000 rpm for 30 seconds.

8. Dispose half of the clear liquid in the centrifuge tube. Mix and levitate the bacteria on the bottom of the tube with a micropipette.

9. In a sterile environment, transfer the transformed bacteria to prepared agar plates.

Colony PCR Identification

A. Materials

2x PCR mix	10 μL
Forward primer	0.5 μL
Reverse primer	0.5 μL
Template	Desired E. Coli
ddH2O	9 μL

B. Protocol

1. Refer to PCR of amilGFP Gene, Jul. 28, 2021.

2. Notes to this reaction:

a. Primers were delivered in the form of dry powder. Centrifuge primers at 12000 rpm for 5 minutes to deposit powder at the bottom of its tube.

b. Dilute primers to a concentration of 10 nmol/ml.

c. Mix the 2x PCR mix, R.primer, Templalate, ddH2O as master mix.

C. Reacting conditions

98 °C	10 minutes	1 cycle
98 °C	15 seconds	26 cycles
55 °C	15 seconds
72 °C	30 seconds

72 °C

5 minutes

1 cycle

4 °C

Infinite

N/A

LB Culture Medium Preperation

Formula

Yeast extract	5 g/L
Tryptone	10 g/L
NaCl	10 g/L
Agar*	1.5% of total mass
ddH2O	Desired volume

*Agar is only necessary when LB culture medium is prepared as solid agar gel.

Sterilization

Sterilize at 121 °C for 30 minutes.

KANA antibiotics

Concentration of KANA antibiotics in solution is 50 mg/μL. Functional concentration of KANA antibiotics is 50 μg/μL.

E. coli Cultivation

A. Protocol

1. Clean workstation with 75% ethanol and work near a lit alcohol lamp in steril conditions.

2. Prepare LB culture medium without KANA antibiotics.

3. Prepare three lines of E. coli with KANA resistance and ARSA, ARSD, or ARSR plasmids depending on the cell line.

4. Add KANA antibiotics to LB culture medium in a ratio of 1 μL KANA antibiotics : 1 mL LB culture medium.

5. Prepare three marked 15 mL centrifure tubes. Distribute 4 mL LB culture medium to each tube.

6. Transfer 10 μL of bacterial solution to its corresponding tube.

7. Cultivate in a shaker in 37 °C at 220 rpm.

Preparation of Agar Plates

A. Protocol

1. Heat the pre-prepared solid LB culture medium in a microwave until the solid completely melts.

2. Add KANA antibiotics in a ratio of 1 μL KANA antibiotics : 1000 μL culture medium. Mix the solutions throughly.

3. In a sterile environment, carefully pour approximately 20-30 mL culture medium to each empty plate.

4. Tilt the plate genly to cover the plate completely with culture medium. Set the plates still and wait for culture medium to cool and solidify.

Preservation of Transformed E. coli

A. Materials

Glycerol	900 µL
LB culture medium with E.coli	900 µL

B. Protocol

1. Sterilize work station and work near a lit alcohol burner. Prepare and mark 2 mL centrifuge tubes.

2. Add 900 µL glycerol to a 2 mL centrifuge tube.

3. Add 900 µL LB culture medium with E.coli to its corresponding tube.

4. Apply laboratory film to seal every centrifuge tube.

5. Store at -20˚C.

6. For further preservation, transfer tubes to be stored at -80˚C after a period of time.

LB Culture Medium with Phosphate Preparation

A. Materials (LB culture medium)

Refer to LB Culture Medium Preparation

B. Materials (phosphate solution)

Na2HPO4.12H2O

25 mg

NaH2PO4.2H2O

13 mg

ddH2O

200 mL

C. Protocol

Weigh 25 mg Na2HPO4 and dissove in 100 mL ddH2O.
Weigh 13 mg NaH2PO4 and dissolve in 100 mL ddH2O.
Sterilize solutions at 121˚C for 30 minutes in a sterilization pot.

Function Test Attempt 1

A. Protocol

1. Sterilize work station and work near a lit alcohol burner.

2. Prepare marked 15 mL test tubes for testing.

3. Distribute 4 mL LB culture medium without KANA antibiotics to each teast tube.

4. Add 40 µL E.coli to its corresponding tube.

5. Cultivate at 37˚C for 2 hours on a shake bed.

6. Dilute NaH2PO4 to 10 mg/L.

7. Add NaH2PO4 to each test tube depending on the testing concentration.

B. NaH2PO4 concentrations

0 µg/L

10 µg/L

20 µg/L

50 µg/L

100 µg/L

150 µg/L

NaH2PO4

(10 mg/L)

0 µL

4 µL

8 µL

NaH2PO4

2 µL

4 µL

6 µL

(100 mg/L)

Function Test Attempt 2

A. Protocol

1. Harvest 3 mL E.coli bacterial solution. Centrifuge at 12000 rpm for 30 seconds to deposit bacterial at the button of the tube. Dispose of the clear liquidon top.

2. Prepare new 15 mL test tubes. Add LB culture medium depending on the target concentration of phosphate.

3. Transfor a moderate amount ofLB culture medium from the test tube to the centrifuge tube.

4. Levitate the bacteria in the centrifuge tube. Transfer the bacterial solution back to test tube.

5. Add phosphate solution to the test tube, depending on the concentration.

6. Cultivate at 37˚C and 220 rpm for 2 hours.

B. Solution amounts

0 mg/L

1 mg/L

10 mg/L

20 mg/L

50 mg/L

100 mg/L

NaH2PO4

(100 mg/L)

0 µL

40 µL

400 µL

800 µL

2000 µL

4000 µL

LB culture medium

4000 µL

3960 µL

3600 µL

3200 µL

2000 µL

0 µL

Function Test Attempt 3

A. Protocol

Refer to Function Test Attempt 2.

B. Notes:

C2H12AsNaO2 is used instead of NaH2PO4.

C. C2H12AsNaO2 amounts

Same as the amounts of NaH2PO4 in Function Test Attempt 2.

To be continued....

Loading buffer & DNA dye	1 μL
PCR product	5 μL
TAE buffer	200 mL
Agarose	2 g

Team:Shanghai United/Experiment