① Gel purification using SanPrep Column DNA Gel Extraction Kit

1. Excise the DNA fragment from the others with Gel electrophoresis as much as possible, cut off the agar gel block containing the target DNA fragment with a clean scalpel, and add it into 1.5 ml centrifugal tube and weight.

2. Add 300 μl Buffer B2 per 100 mg gel slice (Gel concentration ≤ 1%) into the tube. If the block is less than 100 mg then add water to 100 mg.

3. Place the centrifuge tube in a 50℃ water bath for 5-10 min, until the gel slice has completely dissolved.

4. (Optional steps) When the target fragment <500 bp, add 1/3 of the volume of BufferB2 used with isopropanol and mix. When the target fragment >500 bp, this step can be omitted and proceed directly to step 5.

5. Drawn the dissolved solution into the SanPrep column and centrifuge at 8,000 x g for 30 sec. Pour off the liquid in the collection tube and put the SanPrep column into the same collection tube.

6. Add 300 μl Buffer B2 into SanPrep column and centrifuge at 9,000 x g for 30 sec. Pour off the liquid in the collection tube and put the SanPrep column into the same collection tube.

7. Add 500 μl Wash Solution into SanPrep column and centrifuge at 9,000 x g for 30sec. Pour off the liquid in the collection tube and put the SanPrep column into the same collection tube.

8. Repeat step 7 once.

9. Discard flow-through and place the SanPrep column back into the same tube. Centrifuge the column for an additional 1 min. IMPORTANT: This step must never be omitted, otherwise the residual ethanol will severely interfere with yield and subsequent experiments.

10. Add 15-40 μl ddH2O to the center of the SanPrep membrane, let the column stand for 1-2 min, and then centrifuge for 60 s at 9,000 x g. Apply the product to the center of the SanPrep membrane,let the column stand for 1-2 min, and centrifuge for 60 s at 9,000 x g, then store the resulting DNA at -20℃ or use it for subsequent experiments.

② PCR product purification using SanPrep Column PCR Product Purification Kit

1. Apply the PCR sample or the enzymatic reaction mixture to a clean 1.5 ml centrifuge tube. Add 5 volumes of Buffer B3 to this centrifuge tube and then mix. Before first use, check that the appropriate amount of isopropanol has been added. If the sample volume is insufficient, add TE or water to 100 μl, then add 500 μl of Buffer B3 to the sample.

2. Apply the mixture all to the SanPrep column and centrifuge for 30 s at 8,000 x g. Discard flow-through. Place the SanPrep column back into the same tube. If the total volume of the mixture is greater than 750 μl, use 750 μl per time.

3. To wash, add 500 μl Wash Solution to the SanPrep column and centrifuge for 30 s at 9,000 x g. Discard flow-through. Place the SanPrep column back into the same tube. Before first using the Wash Solution, check that correct amount of absolute ethanol has been added.

4. Repeat the step 3 again.

5. Discard flow-through and place the SanPrep column back into the same tube. Centrifuge the column for an additional 1 min. IMPORTANT: This step must never be omitted, otherwise the residual ethanol will severely interfere with yield and subsequent experiments.

6. Add 15-40 μl ddH2O to the center of the SanPrep membrane, let the column stand for 1-2 min, and then centrifuge for 60 s at 9,000 x g. Apply the product to the center of the SanPrep membrane,let the column stand for 1-2 min, and centrifuge for 60 s at 9,000 x g, then store the resulting DNA at -20℃ or use it for subsequent experiments.

③ Plasmid construction using ClonExpress® UItra One Step Cloning Kit

Incubate samples in a thermocycler at 50°C for 30 minutes. Following incubation, store samples on ice for subsequent transformation or at –20°C

④ Making Electrocompetent E. coli Cells

1. Grow an overnight tube culture of E. coli in LB medium.

2. Prepare 50 ml of fresh LB medium in a 250 ml flask for E. coli.

3. Start the flask culture with 500 μl of the overnight tube culture.

4. Grow the cells for approximately 2-3 hours, until they reach OD600 of ~0.6.

5. Transfer the cells to 50 ml Falcon conical tube. Then put the tube into ice for 15 minutes.

6. Pellet the cells by centrifugation for 4 minutes at 8,000 RPM. Remove promptly and pour off supernatant.

7. Wash by adding 25 ml of chilled 10% glycerol to each tube, then resuspend the pellet gently. Centrifuge for 4 minutes at 8,000 RPM. Remove promptly and pour off supernatant.

8. Repeat step 7

9. Resuspend in approximately 250 μl of 10% glycerol to make a 100x concentration of the initial culture.

10. Divide into 50 μl aliquots in 1.5 ml tubes. Freeze at -80 degree or proceed directly to electroporation.

⑤ Electroporation

1. Place SOB recovery medium at room temperature for 1 hour.

2. Place electroporation cuvettes (1 mm), desalted DNA, and Electrocompetent E. coli Cells on ice.

3. Carefully transfer the cell/DNA mix into a chilled cuvette without introducing bubbles and make sure that the cells deposit across the bottom of the cuvette. Electroporate using the following conditions for Bio-Rad GenePulser electroporators: 1.8 kV, 100 Ω, and 25 μF.

4. Immediately add 950 µl of 37°C SOB to the cuvette, gently mix up and down twice, then transfer to the 1.5 ml tube.

5. Shake vigorously (200 rpm) at 37°C for 1 hour.

6. Dilute the cells as appropriate then spread 100-200 μl cells onto a pre-warmed selective plate.

7. Incubate plates 8 hours to overnight at 37°C.

⑥ Plasmid DNA Purification Using SanPrep Column Plasmid Mini-Preps Kit

1. Inocolate the target strain in the medium containing the appropriate selective antibiotic. Incubate for approximately 12-16 h at 37℃with vigorous shaking.

2. For High-copy plasmids, take 1.5-5 ml of bacterial solution, then harvest the bacterial cells by centrifugation at 6000 x g for 15 min at room temperature. Discard the medium.

3. Resuspend the bacterial pellet in 250 μl of Buffer P1.

4. Add 250 μl of Buffer P2, mix thoroughly by vigorously inverting the sealed tube 5−10 times, and incubate at room temperature for 2-4 min.

5. Add 350 μl of Buffer P3, mix immediately and thoroughly by vigorously inverting 5–10 times.

6. Centrifuge at maximum speed(≥12,000 x g) in a centrifuge for 5-10 min. Remove the supernatant containing plasmid DNA promptly, place the SanPrep column in the same collection tube.

7. OPTIONAL: Add 500 μl Buffer DW1, then centrifuge for 30 s at 9,000 x g. Remove the supernatant containing plasmid DNA promptly, place the SanPrep column in the same collection tube.

8. Add 500 μl Wash Solution to SanPrep column, then centrifuge for 30 s at 9,000 x g. Remove the supernatant containing plasmid DNA promptly, place the SanPrep column in the same collection tube.

9. Repeat step 8 once.

10. Place the empty SanPrep column and collection tube to centrifuge, then centrifuge 1 min at 9,000 x g.

11. Add 15-40 μl ddH2O to the center of the SanPrep membrane, let the column stand for 1-2 min, and then centrifuge for 60 s at 9,000 x g. Apply the product to the center of the SanPrep membrane,let the column stand for 1-2 min, and centrifuge for 60 s at 9,000 x g, then store the resulting DNA at -20℃ or use it for subsequent experiments.