Experiments

1) LB media, Liquid (g/L)

1. 10.0 g Peptone
2. 5.0 g Yeast Extract
3. 10.0 g NaCl
4. Sterilization for 20 min under 1×10^5 Pa

2) LB media, Powder (g/L)

1. 15.0 g Agar powder
2. 10.0 g Peptone
3. 5.0 g Yeast Extract
4. 10.0 g NaCl
5. Sterilization for 20 min under 1×10^5 Pa Amp+ antibiotics

1) Add the following reagents to PCR tubes to form a PCR reaction system.

2) PCR Program:

1. 95 ℃ 3 min;
2. 98 ℃ 15 s
3. 61 ℃ 15 s
4. 68 ℃ 30 s; 2-4 for 35 cycles
5. 68 ℃ 5 min; 4℃ ∞

3) Agarose gel electrophoresis

• Add 0.8 g agarose regular in 80 mL TAE buffer. Dissolve it under high temperature (microwave for about 2 minutes until mixture turns transparent), wait till it cools down. Then add 8 mL nucleic acid dye (10000 x)
• Add 2× DNA loading buffer into PCR product.
• Put the agarose in the electrophoresis tank, and remove the comb. Add 20 µL of each solution into each well in the agarose gel.
• Gel electrophoresis: 120 V, 20 min.
• Use UV to visualize the DNA strand, and save the figure.

4) Gel extraction

• Cut down the gel which containing the target fragment and calculate the weight difference in the EP tube from adding the gel by weighing the tube before and after adding the gel.
• Add Buffer B2 at a rate of 300-600 μL per 100 mg of agarose, depending on the weight and concentration of the gel.
• Place the centrifuge tube in a 50°C water bath for 5-10 min and mix occasionally until the gel block is completely dissolved.
• Transfer the entire dissolved solution to the adsorbent column and centrifuge at 8,000×g for 30 sec. Pour off the liquid from the collection tube and place the column in the same collection tube.
• Add 500 μL Wash Solution to the column, centrifuge at 9,000×g for 30 sec and pour off the liquid from the collection tube and place the column in the same collection tube.
• Repeat step 5 once.
• Place the empty column and collection tube into a centrifuge and centrifuge at 9,000×g for 1 min. Add 15-40 μL of Elution Buffer to the center of the adsorbent membrane, leave at room temperature for 1-2 min, and centrifuge at 9,000×g for min. Store the resulting DNA solution at -20°C or use it for subsequent experiments.

1) Culture of strain

• Inoculate the strain into the LB medium.
• Incubate the bacteria at 37℃ and 220 rpm, overnight.

2) Extract plasmid

Use Plasmid Mini-Preps Kit:

• Add RNase A to Buffer SP1
• Add absolute ethanol to Washing solution
• Take 1.5mL of overnight cultured bacterial solution, centrifuge at 8000×g for 2 minutes, and pour off the medium.
• Add 250 μl Buffer SP1 to the tube to suspend the cells thoroughly
• Add 250 μl of Buffer SP2, gently invert the centrifuge tube 10 times immediately. Standing at room temperature for 10 minutes.
• Add 350 μl Buffer SP3 and gently invert the centrifuge tube 10 times
• Centrifuge at 12000 ×g for 8 minutes. Transfer the supernatant to the adsorption column, centrifuge at 8000Xg for 30 seconds, and pour off the liquid in the collection tube
• Add 500 mL of wash solution, centrifuge at 9000×g for 30 seconds, and pour off the liquid in the tube (repeat once)
• Centrifuge the empty adsorption column at 9000 ×g for 1 minute
• Put the adsorption column into a new 1.5mL centrifuge tube, add 100 mL of Elution Buffer to the adsorption membrane, stand at room temperature for 1 minute, and centrifuge for 1 minute.

3) Double digestion of plasmids

4) Agarose gel electrophoresis and gel extraction

5) Homologous recombination to construct the vectors

Add gene fragments and the vector in a ratio of 3:1. Use 2× Hieff Clone® Universal One Step Cloning Kit to conduct homologous recombination.

6) Transformation and cell culture

• Transform into DH5α competent, Place on ice for 25 minutes Heat shock (42℃, 90 seconds), Place on ice for 2 minutes,Add 500 µL of LB liquid medium.
• Place in an oven/shaker at 37 ℃, 1 hour
• Centrifuge at 3000 rpm for 2-3 minutes
• Discard the supernatant, leaving 20 μL - 50 μL LB liquid medium, and mix it with the bacterial solution.
• Add the bacterial solution to the solid medium plate with ampicillin antibiotics, spread the plate with a coating bar, place it in an oven at 37 ℃, and cultivate for 12-16 hours.

1) Configure PCR reaction system