LB Liquid Medium（1L）

LB Solid Medium（1L）

DH5α

1. Thaw competent cells on wet ice. Place the required number of 1.5 mL eppendorfs on wet ice, then make 100 µL aliquots of competent cells in the chilled 1.5 mL microcentrifuge tubes.

2. Add 10 µL of sample DNA directly into a tube of competent cells. Mix well by gently flicking the tube several times.

3. Incubate the cells on ice for 30 min.

4. Heat-shock the cells for exactly 45 sec in a 42℃ water bath.

5. Incubate the cells on ice for 2 min.

6. Add 900 µL of LB Liquid Medium.

7. Shake the tube at 200 rpm for 1 h at 37℃.

8. Centrifugate eppendorfs at 12000 rpm for 3 min and discard 900 µL supernatant. Resuspend cells with the remaining liquid.

9. Spread plates with bacterial suspension and incubate for the night.

BL21(DE3)

1. Thaw competent cells on wet ice. Place the required number of 1.5 mL eppendorfs on wet ice, then make 100 µL aliquots of competent cells in the chilled 1.5 mL microcentrifuge tubes.

2. Add 10 µL of sample DNA directly into a tube of competent cells. Mix well by gently flicking the tube several times.

3. Incubate the cells on ice for 30 min.

4. Heat-shock the cells for exactly 90 sec in a 42℃ water bath.

5. Incubate the cells on ice for 2 min.

6. Add 900 µL of LB Liquid Medium.

7. Shake the tube at 200 rpm for 1 h at 37℃.

8. Centrifugate eppendorfs at 12000 rpm for 3 min and discard 900 µL supernatant. Resuspend cells with the remaining liquid.

9. Spread plates with bacterial suspension and incubate for the night.

(According to FastPure® Plasmid Mini Kit)

1. Transfer 1 - 5 mL of overnight (12 - 16 h) culture to a centrifuge tube, and centrifuge at 10,000 rpm (11,500 × g) for 1 min. Discard the culture medium and place the tube inverted on blotting paper to drain the liquid.

2. Add 250 μL of Buffer P1 (make sure that RNase A has been added) to the centrifuge tube containing the precipitated bacterial cells, and mix thoroughly by pipetting or vortexing.

3. Add 250 μL of Buffer P2 to the mixture from Step 2. Mix by gently inverting the tube 8 - 10 times to completely lyse the cells.

4. Add 350 μL of Buffer P3 to the mixture from Step 3. Immediately invert the tube gently 8 - 10 times to fully neutralize Buffer P2. At this time, white flocculent precipitates should form. Centrifuge at 12,000 rpm (13,400 × g) for 10 min.

5. Place FastPure DNA Mini Columns into a Collection Tube 2 mL. Carefully transfer the supernatant from Step 4 to the FastPure DNA Mini Columns with a pipette, taking care not to disturb the precipitates. Centrifuge at 12,000 rpm (13,400 × g) for 30 - 60 sec. Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.

6. Add 600 μL of Buffer PW2 (make sure that absolute ethanol has been added) to the FastPure DNA Mini Columns. Centrifuge at 12,000 rpm (13,400 × g) for 30 - 60 sec. Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.

7. Add 600 μL of Buffer PW2 (make sure that absolute ethanol has been added) to the FastPure DNA Mini Columns. Centrifuge at 12,000 rpm (13,400 × g) for 30 - 60 sec. Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.

8. Place the FastPure DNA Mini Columns back into the Collection Tube. Centrifuge the empty column at 12,000 rpm (13,400 × g) for 1 min to completely remove the residual wash buffer.

9. Place the FastPure DNA Mini Columns in a new sterile 1.5 mL centrifuge tube. Add 30 - 100 μL of Elution Buffer to the center of the spin column membrane. Leave the system at room temperature for 2 min, and centrifuge at 12,000 rpm (13,400 × g) for 1 min to elute the DNA.

10. Discard the FastPure DNA Mini Columns and store the extracted DNA at -20°C to prevent degradation.

1. Place the gel tray in the appropriate position in the gel cartridge and place the comb in the correct position.

2. Measure 0.5 g agarose, put it in a 250 mL Erlenmeyer flask, add 50 mL 1 × TAE buffer and mix, then put the Erlenmeyer flask in the oven and heat to boil until the agarose is completely dissolved.

3. Add 5 μL GelRed to the solution.

4. Pour the solution into the gel casting tray.

5. After the gel cools to solid, pull out the comb.

6. Place the gel in the electrophoresis chamber with enough TAE buffer.

7. Add 10 × loading buffer to the sample and mix, then transfer the mixture to the well on the gel with a pipette.

8. Power on, run at 120 V for 30 min.

(According to FastPure® Gel DNA Extraction Mini Kit)

1. After DNA electrophoresis for fractionating DNA fragments, excise the single stripe of DNA fragment from the agarose gel under UV light. It is recommended to use absorbent paper to absorb apparent moisture of gel and minimize the size of the gel slice by removing extra agarose as possible as you can. Weigh the gel slice (without microcentrifuge tube) to calculate its volume: The volume of 100 mg gel slice is approximately 100 μL, assuming the density is 1 g/mL.

2. Add equal volume Buffer GDP, incubate at 50 ~ 55℃ for 7 - 10 min (according to the gel size, adjust incubation time until the gel completely dissolved). Invert the tube 2 times during incubation.

3. Centrifuge briefly to bring the sample to the bottom of the tube, insert the FastPure DNA Mini Columns-G into the Collection Tubes 2 mL, carefully transfer the solution maximally of 700 μL once a time to the filtration columns, centrifuge at 12,000 rpm (13,800 × g) for 30 - 60 sec.

4. Discard the filtrate and add 300 μL of Buffer GDP to the column, incubate at room temperature for 1 min, centrifuge at 12,000 rpm (13,800 × g) for 30 - 60 sec.

5. Discard the filtrate and add 700 μL of Buffer GW (check if absolute ethanol has been added in advance!) to the column, centrifuge at 12,000 rpm (13,800 × g) for 30 - 60 sec.

6. Discard the filtrate and add 700 μL of Buffer GW (check if absolute ethanol has been added in advance!) to the column, centrifuge at 12,000 rpm (13,800 × g) for 30 - 60 sec.

7.Discard the filtrate and centrifuge the empty column at 12,000 rpm (13,800 × g) for 2 min.

8. Insert the column into a clean 1.5 mL microcentrifuge tube, add 20 - 30 μL of Elution Buffer to the center of the column membrane, incubate for 2 min, and then centrifuge at 12,000 rpm (13,800 × g) for 1 min. Discard the column, store the obtained DNA at -20℃.

1. Keep all components on ice during the experiment. Thaw, mix, and briefly centrifuge each fraction before use. After use, put them back in -20 ℃.

2. Gently centrifuge to collect the liquid at the bottom of the tube.

3. Transfer the PCR tube to the PCR machine, set the parameters and start the thermal cycle.

Reaction System

Reaction Program

1. The amount of DNA can be roughly calculated according to the above formula. Dilute the vector and insert at an appropriate ratio to ensure the accuracy of pipetting before preparing the recombination reaction system, and the amount of each component is not less than 1 µL.

For ClonExpress II recombination reaction system, the optimal amount of vector required is 0.03 pmol, the optimal amount of insert required is 0.06 pmol (the molar radio of vector to insert is 1:2). These masses can be roughly calculated according to the following formula:

The optimal mass of vector required = [0.02 × number of base pairs] ng (0.03 pmol)

The optimal mass of insert required = [0.04 × number of base pairs] ng (0.06 pmol)

2. Prepare the following reaction on ice:

3. Gently pipette up and down for several times to mix thoroughly (DO NOT VORTEX!). Briefly centrifuge to collect the reaction solution to the bottom of the tube.

4. Incubate at 37℃ for 30 min and immediately chill the tube at 4℃ or on ice.

1. Bacterial Culture Preparation

Inoculate bacterial culture by adding 1:100 of the overnight-grown bacteria to liquid LB medium. Add appropriate antibiotics to the culture at a final concentration of 1:1000 (Ampicillin final concentration: 100 mg/L or Kanamycin final concentration: 50 mg/L). Incubate the culture at 37°C by shaking 200 rpm for 2 h.

2. OD Measurement

Use a spectrophotometer to monitor the optical density (OD₆₀₀) of the culture. Continue shaking until the OD₆₀₀ reaches 0.6-0.8, indicating the cells are in the mid-log phase, which is the optimal point for induction.

3. Induction Preparation

Once the culture reaches the desired OD₆₀₀, transfer the Erlenmeyer flasks to an ice-water bath and allow the culture to cool rapidly for 5 min. Ensure that the solution has completely cooled before proceeding to induction.

4. Induction

Add IPTG at a final concentration of 1:2000(0.5 M IPTG) to the culture to induce protein expression. Shake the culture at 150 rpm at 16°C for 20 h to allow the expression of the target protein.

Note: For each protein, prepare two cultures—one without IPTG as the control, and one with IPTG for protein induction.

5. After Induction

After the overnight incubation, harvest the cells by centrifugation or proceed to downstream protein purification.

1. Preparation of Buffer:

Dissolve one pack of MOPS-SDS Running Buffer Powder in 1 L of deionized water to prepare a 1× electrophoresis buffer.

Note: It is recommended that the Running Buffer should not be reused more than three times.

2. Preparation of Precast Gel:

Remove the precast gel from its packaging and peel off the sealing strip from the bottom of the gel cassette.

Following the direction indicated by the arrow, gently and parallelly push the comb out of the gel cassette to avoid leaving any residue in the wells.

3. Installation of Precast Gel:

Install the precast gel into the electrophoresis tank.

4. Filling the Electrophoresis Tank:

Pour sufficient MOPS-SDS Running Buffer into the inner chamber of the electrophoresis tank, ensuring that the buffer covers the wells by 5-7 mm. Add the same buffer to the outer chamber.

For optimal results, the buffer level in the outer chamber should be slightly lower than that in the inner chamber, ensuring that the buffer does not overflow the gel cassette.

Using a syringe or another appropriate tool, gently rinse the sample wells with 1× electrophoresis buffer to remove any bubbles or residual storage buffer.

5. Loading Samples and Running the Gel:

Load the protein samples and protein marker (Trelief® Prestained Protein Ladder, Catalog No.: TSP021) into the wells.

Run the gel at a recommended voltage of 160 V, not exceeding 180 V for 30 min.

6. Gel Storage:

After electrophoresis, carefully remove the gel cassette from the electrophoresis tank.

Insert an appropriate prying tool into the gap between the gel plates.

Gently pry at the top, middle, and bottom of the gel cassette until the plates are completely separated.

Once the plates are open, transfer the gel to water for subsequent steps.

7. Membrane Transfer:

Prepare filter paper (10*7.5 cm) and PVDF membrane (9*7.2 cm).

Wet the PVDF membrane in methanol for 1-2 minutes.

Prepare a “sandwich” setup for transfer: black plate → sponge → filter paper → gel →PVDF membrane → filter paper → sponge.

Transfer at 100V for 90 minutes with constant voltage.

8. Blocking:

Place the PVDF membrane into a container and add the blocking solution. Shake for 1 hour to block nonspecific binding sites.

9. Primary Antibody Incubation:

After blocking, discard the blocking solution and add 20ml of the primary antibody (diluted 1:100 in TBST) into each container. Shake for 1 hour at room temperature.

Wash the membranes with 1xTBST for 5 minutes three times to remove unbound primary antibodies.

10. Secondary Antibody Incubation:

Wash the membranes with 1xTBST for 5 minutes three times.

11.Exposure:

Pour 3ml of the ECL mixture onto the plastic wrap in separate spots.

Using tweezers, submerge the PVDF membrane in the ECL mixture, following this sequence: front side down, back side down, front side down again, leaving it in each position for 1 minute.

Align the film with the membrane inside the exposure box and start the timer. Set the exposure time to different intervals (e.g., 30 seconds, 1 minute, 5 minutes, 10 minutes).

After each exposure time, retrieve the film and process it.

Once outside the dark room, label the film with the date, sample number, and exposure time.

Use the protein marker on the PVDF membrane as a reference to mark the bands on the film.

12. Ponceau Staining:

Stain the membrane with Ponceau S staining solution until red protein bands appear. Wash the membrane with water to remove excess stain.