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