Complete Workflow for Recombinant Plasmid Construction

Experimental Procedures

I. Preparation of LB Medium

LB medium is commonly used for bacterial culture, particularly for the maintenance and propagation of E. coli in molecular biology experiments. Tryptone and yeast extract provide nitrogen sources, vitamins, and growth factors; NaCl helps maintain osmotic balance; glucose provides a carbon source; and agar serves as the solidifying agent.

Reagents

• Tryptone

• Yeast Extract

• NaCl

• Agar

Experimental Procedure

1. LB Solid Medium Formula

For 100 mL of LB solid medium:

Tryptone readily absorbs moisture, so work quickly when weighing it. To prevent cross-contamination between reagents, use a dedicated spatula for each reagent. Alternatively, thoroughly wash and dry the spatula before weighing a different reagent. Make sure reagent bottle caps are returned to the correct bottles.

2. LB Liquid Medium

For LB liquid medium, use the same formulation as the solid medium, except that agar is omitted.

3. Sealing and Labeling

Cover the bottle opening with paper and secure it with a rubber band. Label the bottle with the medium name, group, and preparation date.

4. Sterilization

Autoclave the prepared medium at 1.05 kg/cm² and 121.3°C for 20 minutes. If immediate sterilization is not possible, temporarily store the medium at 4°C.

After sterilization, place the Erlenmeyer flask in a drying oven. Once dry, store the medium at 4°C.

5. Preparation of LB Agar Plates

① Preparation

Prepare 100 mL of LB solid medium according to the formulation above.

② Addition of Antibiotics

Using ampicillin (Amp) resistance as an example, heat the solidified medium in a microwave until completely melted. Then place it in a 55°C water bath. Once the medium has cooled to approximately 55°C, add 100 μL of ampicillin and mix thoroughly. Avoid adding antibiotics at excessively high temperatures, as heat may reduce antibiotic activity.

③ Pouring Plates

Generally, approximately 20 mL of medium is used per plate. Pour the medium into sterile Petri dishes and leave the lids partially open under UV light for approximately 15–20 minutes.

④ Storage

Invert the plates and store them at 4°C. Use the plates within one month.

II. PCR Amplification of the Target Gene

Reagents

• Sterile water

• High-fidelity DNA polymerase (2×)

• Forward primer

• Reverse primer

• Template DNA (plasmid)

Procedure

Preheat the PCR instrument before starting the reaction.

1. Prepare the PCR Reaction

Add the following components sequentially to a 0.5 mL PCR tube to prepare a 20 μL reaction:

2. PCR Amplification

Place the PCR reaction mixture into the PCR instrument.

3. PCR Program

Set the amplification program according to the manufacturer's instructions for the high-fidelity DNA polymerase being used.

Example program:

Initial denaturation: 94°C for 5 min

Then perform 30 cycles of:

• 94°C: denaturation, 30 s

• 55°C: annealing, 1 min

• 72°C: extension, 30 s

The annealing temperature should be optimized according to the primers used. The extension time should be adjusted according to the length of the target fragment.

After the final cycle, perform a final extension at 72°C for 7 min, followed by cooling at 4°C.

4. PCR Product Detection

Analyze the PCR products by electrophoresis on a 1.5% agarose gel.

Note: When adding components to the PCR tube, the reagents can be dispensed onto the side of the tube first and mixed at the end. This can help save time and reduce pipette tip consumption.

III. PCR Product Recovery and Purification

1. Gel Dissolution

Briefly centrifuge the PCR products and perform agarose gel electrophoresis to identify the target band. Excise the desired DNA band and transfer the gel slice into a clean 1.5 mL microcentrifuge tube.

Add 330 μL of gel dissolution buffer, corresponding to approximately two-thirds of the gel slice volume. Incubate at 60°C for approximately 30 minutes until the gel is completely dissolved.

Note: Minimize exposure of the DNA to UV light during gel excision to prevent DNA damage.

2. Column Equilibration

Place the spin column into a collection tube. Add 100 μL of BL buffer and centrifuge at 12,000 rpm for 30 s. Discard the flow-through.

3. DNA Binding

Transfer the dissolved gel solution from Step 1 to the spin column. Allow it to stand for 2 minutes to facilitate DNA binding to the column membrane.

Centrifuge at 12,000 rpm for 30 s and discard the flow-through.

4. Washing

Add 450 μL of PW buffer. Centrifuge at 12,000 rpm for 30 s and discard the flow-through.

5. Repeat the Washing Step

Repeat Step 4 once.

Note: Steps 2–5 can also be performed using a vacuum filtration system.

6. Dry Spin

Centrifuge at 12,000 rpm for approximately 2 minutes. Discard the collection tube and transfer the spin column to a new 1.5 mL microcentrifuge tube.

Open the cap and allow the column to air-dry for 5–10 minutes to remove residual ethanol.

7. DNA Elution

Apply 30 μL of TB buffer directly to the center of the membrane without touching it. Incubate at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 30 s.

For faint DNA bands, a smaller elution volume of approximately 18–20 μL can be used.

8. Labeling and Storage

Discard the spin column and label the tube with the appropriate sample information. Store the purified DNA at −20°C.

IV. Restriction Digestion of the Vector

Reagents

• Cut Buffer

• Restriction enzyme I (EcoRI)

• Restriction enzyme II (BamHI)

• Plasmid DNA

• Sterile water

Procedure

1. Prepare the Water Bath

Prepare the water bath before beginning the reaction.

2. Prepare the Restriction Digestion Reaction

Add the following components to a 0.5 mL PCR tube:

Note: The volumes of the components shown in bold remain constant and should not be changed when adjusting the total reaction volume.

3. Restriction Digestion

Incubate at 37°C for 30 minutes.

4. Product Detection

Analyze the double-digested products by agarose gel electrophoresis.

Note on Buffer Selection: The selected buffer must be compatible with both restriction enzymes. Refer to the manufacturer's official documentation for detailed information regarding enzyme compatibility and reaction conditions.

V. Recombinant Reaction — Fragment and Vector Assembly

Using a two-fragment assembly as an example, prepare a 10 μL recombinant reaction:

Incubate at 50°C for 30 minutes, or follow the manufacturer's instructions for the recombination enzyme being used.

VI. Transformation of the Recombinant Product

1. Transformation

① Addition of Recombinant Product

Add the entire 10 μL recombinant reaction to competent cells and incubate on ice for 40 minutes.

② Heat Shock

Heat-shock the competent cells at 42°C for 90 seconds to facilitate plasmid uptake.

③ Recovery on Ice

Immediately place the cells on ice for 5 minutes.

④ Recovery Culture

Add 200 μL of antibiotic-free LB liquid medium and incubate at 37°C with shaking for 1 hour.

2. Plate the Transformed Cells

Spread the recovered culture onto LB agar plates containing ampicillin (Amp) and incubate overnight.

3. Pick Individual Colonies

On the following day, select individual colonies and inoculate them into 5 mL of LB liquid medium containing ampicillin.

For example, add 5 μL of ampicillin to 5 mL of LB medium, then use a sterile pipette tip to transfer the selected colony into the culture medium.

Incubate overnight with shaking at 38°C. The resulting bacterial culture can be used for plasmid extraction the following day.

VII. Plasmid Extraction

1. E. coli Culture

Pick a single colony from the LB agar plate and inoculate it into 5 mL of LB liquid medium containing the appropriate antibiotic. Incubate overnight at 37°C with shaking.

Note: Avoid using excessive culture volumes. Overgrowth can result in incomplete bacterial lysis and may negatively affect plasmid purity during purification.

2. Harvest the Bacterial Cells

Transfer 5 mL of bacterial culture into a 5 mL centrifuge tube. Centrifuge at 10,000 × g for 1 minute and discard the supernatant.

Note: The original protocol uses rpm as an alternative when the laboratory centrifuge cannot reach the specified RCF. RCF (×g) is the preferred standard for reporting centrifugation conditions because rpm depends on rotor radius. The same rpm does not necessarily produce the same centrifugal force on different centrifuges, whereas the same RCF provides a more comparable centrifugal force.

3. Resuspend the Cell Pellet

Add 250 μL of Solution I containing RNase A, which should be added according to the manufacturer's instructions before use. Store the prepared Solution I at 4°C.

Thoroughly resuspend the bacterial pellet.

Note: Make sure no visible bacterial clumps remain. A vortex mixer can be used to facilitate complete resuspension.

4. Cell Lysis

Add 250 μL of Solution II to lyse the cells. Pre-equilibrate Solution II at 37°C if required by the reagent protocol.

Gently invert the tube 5–6 times to mix thoroughly until a clear lysate is formed.

Note: Do not extend this step beyond the time recommended by the plasmid extraction kit manufacturer.

5. Neutralization

Add 350 μL of Solution III and gently invert the tube 5–6 times until a compact precipitate forms.

6. Clarification

Transfer the lysate to a 1.5 mL microcentrifuge tube and centrifuge at 13,000 × g for 10 minutes.

7. Column Binding

Transfer the clarified supernatant to the spin column and centrifuge at 10,000 × g for 1 minute. Discard the flow-through.

8. Wash

Add 500 μL of Buffer HB and centrifuge at 10,000 × g for 1 minute. Discard the flow-through.

9. DNA Wash

Add 700 μL of DNA Wash Buffer containing the specified amount of 100% ethanol added according to the manufacturer's instructions.

Centrifuge at 10,000 × g for 1 minute and discard the flow-through. Repeat this washing step once.

10. Dry Spin

Centrifuge at 13,000 × g for 2 minutes to remove residual wash buffer.

11. Dry the Column

Transfer the spin column to a clean 1.5 mL microcentrifuge tube and allow it to dry for approximately 3 minutes according to the kit instructions.

12. Elution

Add 60 μL of preheated ddH₂O to the center of the spin column membrane. Preheat the water to approximately 60°C if recommended by the extraction protocol.

Allow the column to stand for 2 minutes.

13. Collect the Plasmid DNA

Centrifuge at 13,000 × g for 1 minute to collect the purified plasmid DNA.

14. Determine DNA Concentration

Measure the concentration of the purified plasmid DNA using an appropriate nucleic acid quantification method.

15. Agarose Gel Electrophoresis

Analyze the purified plasmid DNA by electrophoresis on a 1.5% agarose gel.

Note: Label purified plasmid samples with the sample name, preparation date, and other relevant information. Store at −20°C.

VIII. Agarose Gel Electrophoresis

Reagents

• Agarose

• 1× TAE electrophoresis buffer

• DNA stain (SYBR Safe DNA Gel Stain)

• DL2000 DNA Marker

• 10× Loading Buffer

Procedure

1. Preparation of 1.5% Agarose Gel

Prepare either 50 mL of gel for a larger gel or 25 mL for a smaller gel.

For 50 mL of 1.5% agarose gel, weigh 0.75 g of agarose and add it to 50 mL of 1× TAE buffer. Add SYBR Safe DNA Gel Stain according to the manufacturer's recommended concentration.

Mix thoroughly and seal the flask appropriately.

2. Dissolve the Agarose

Heat the agarose solution in a microwave until completely dissolved and homogeneous, taking care to prevent excessive boiling.

3. Cast the Gel

Insert the comb and pour the molten agarose into the gel casting tray.

4. Allow the Gel to Set

Place the gel at room temperature away from light and allow it to solidify for approximately 20 minutes.

5. Load the Samples

① For DNA Detection — Small Wells

② For DNA Recovery — Large Wells

6. Electrophoresis

Run the agarose gel at 120 V for approximately 25 minutes.

7. Detection

Place the gel in the gel documentation system for imaging.

Select:

New → Select Stain (SYBR Safe) → Select Color (SYBR Green) → Deselect Highlight → Detect → Text Annotation → Save

For text annotations, use a font size of 14 and white text.

8. Quality Assessment and Sequencing

Verify that the concentration and expected size of the purified plasmid meet the requirements for sequencing. Submit qualified samples for sequencing and store the remaining samples at −20°C.

Notes

1. When the sample volume is less than 5 μL, add 2 μL of 10× Loading Buffer.

2. When the well width is less than 6 mm, loading approximately 5 μL of sample is generally sufficient to obtain a clear DNA band. If the wells are wider, appropriately increase the amount of DNA marker loaded.

3. For DNA electrophoresis, the purity of the agarose can significantly affect the clarity of DNA bands.

4. Agarose concentration has a major influence on DNA fragment separation. Higher agarose concentrations generally provide better resolution of shorter DNA fragments, whereas lower concentrations are generally more suitable for resolving longer DNA fragments.

5. Use a gel with larger wells when DNA fragments need to be recovered. When electrophoresis is performed only for analytical detection, a gel with smaller wells can be used.

6. The specifications and band sizes of the DNA marker should be confirmed according to the manufacturer's official documentation.