Plasmid Midi Kit-HYC212

Components

Storage

This kit can be stored at ambient temperature for up to 18 months. RNase A can be shipped and stored at room temperature; for long-term storage (over 3 months), keep it at −20~8 °C. Buffer P2 may form precipitates under low temperature. Prior to use, incubate in a 37 °C water bath to fully dissolve all precipitates.

Instructions

Preparations

1. Add 0.5 mL Buffer P1 to RNase A powder, pipette up and down repeatedly to fully dissolve RNase A, then transfer the entire mixture into the bulk Buffer P1 bottle.If RNase A is provided in liquid form, perform a brief centrifugation and transfer all contents into Buffer P1. The prepared Buffer P1 containing RNase A is stable for 6 months when stored at 2–8 °C.
2. SDS in Buffer P2 may precipitate at low temperatures. Incubate in a 37 °C water bath before use to dissolve all precipitates completely.
3. Dilute Buffer PW2 with 4 volumes of absolute ethanol as indicated on the bottle label, and store at ambient temperature.

Procedures

1. Inoculate E. coli strain carrying the target plasmid into a 5–10 mL culture tube with 1 mL LB medium supplemented with the corresponding antibiotic, and incubate at 37 °C with shaking for 6–8 h for primary seed culture.

Cultivation procedure: Under sterile conditions, pick a single colony with a sterile toothpick and transfer it into 1 mL antibiotic-containing LB medium, then shake at 200–300 rpm and 37 °C for 6–8 h. Glycerol stock strains may lose the target vector during storage; streak on agar plates for activation first, then pick a single colony for primary culture.

2. Add 50–75 mL antibiotic-supplemented LB medium into a 250 mL culture flask, inoculate with 0.1% volume of primary seed culture, and incubate at 37 °C with shaking for 12–14 h.

The flask volume should be at least 4–5 times the volume of culture medium. After overnight incubation, assess cell density by OD₆₀₀ measurement. Well-grown cultures in LB medium yield an OD₆₀₀ of 2.0–3.0. 2×YT or TB medium accelerates bacterial overgrowth and inhibits sufficient plasmid replication, so their use is discouraged. If YT or TB medium is unavoidable, adjust the input volume based on bacterial biomass. Biomass reference value = 150; for cultures grown in YT/TB medium with OD₆₀₀ = 10, use 15 mL of culture for high-copy plasmids.

3. Transfer 50–75 mL culture into a suitable centrifuge tube, pellet cells at 4,000–5,000 rpm for 10 min, discard supernatant, and tap the tube briefly on absorbent paper to drain residual liquid.

The maximum DNA binding capacity of each DNA Midi Column is 300 μg. Select the appropriate culture volume according to your plasmid copy number. For low-copy vectors, up to 150 mL LB culture can be processed with doubled lysis reagent volumes: 5 mL Buffer P1, 5 mL Buffer P2 and 7 mL Buffer P3. This kit contains sufficient reagents for processing 150 mL culture.

4. Add 2.5 mL Buffer P1/RNase A mixture to the cell pellet, then vortex at high speed or pipette up and down repeatedly to fully resuspend the bacterial pellet.

Complete resuspension is critical for high DNA yield; no visible cell clumps should remain after resuspension. If clumps persist after vortexing, pipette the suspension repeatedly to disperse aggregates.

5. Add 2.5 mL Buffer P2 to the resuspension, gently invert and rotate the tube 15–20 times, then incubate at room temperature for 3 min with 6–8 gentle inversions during incubation.

Do not vortex during mixing. Complete lysis produces a homogeneous, clear viscous solution. Vortexing causes genomic DNA contamination. If the solution remains turbid after mixing, the cell load is excessive; reduce the culture volume or increase volumes of Buffer P1, P2 and P3 in subsequent runs. When processing 75 mL culture, the lysate will be highly viscous (high-density alkaline lysis format). Perform more inversion cycles and gentle agitation to fully lyse cells and generate a lump-free homogeneous lysate.

6. Add 3.5 mL Buffer P3 to the lysate, invert rapidly 10–15 times immediately to form a flocculent precipitate.

Mix promptly after adding Buffer P3 to avoid localized precipitation. For 75 mL culture processing (high-density alkaline lysis), large compact aggregates will form upon neutralization. Carry out repeated inversion and gentle shaking to break large clumps into smaller fragments, enabling full penetration of Buffer P3 for complete neutralization.

7. Centrifuge the neutralized mixture at 4,000–5,000 rpm for 15 min.

If a high-speed centrifuge is available, transfer the mixture to high-speed tubes and centrifuge at 8,000–12,000 rpm for 10 min. If the supernatant remains cloudy, filter through MagPure mini filters to remove debris.

8. Column equilibration: Fit the DNA Midi Column onto a 15 mL collection tube, load 0.5 mL Buffer BCL onto the column membrane, then centrifuge at 4,000–5,000 rpm for 2 min and set aside for use.
9. Load 4 mL clarified supernatant onto the equilibrated DNA Midi Column, centrifuge at 4,000–5,000 rpm for 2 min, discard flow-through and reattach the column to the collection tube. Repeat this step until all supernatant has passed through the column.
10. Discard flow-through, reattach the column to the collection tube, add 2 mL Buffer PW1 to the column, and centrifuge at 4,000–5,000 rpm for 2 min.
11. Discard flow-through, reattach the column to the collection tube, add 4 mL ethanol-diluted Buffer PW2 to the column, and centrifuge at 5,000 rpm for 10 min.

Avoid touching the column bottom to residual liquid when retrieving the column; re-centrifuge if contamination occurs.

12. Remove the DNA Midi Column and air-dry the membrane at room temperature for 10 min. Discard waste liquid from the collection tube, rinse the tube once with sterile water, discard the rinse liquid and air-dry for subsequent use.

Buffer PW2 contains 80% ethanol for mild disinfection. After discarding flow-through and drying, the collection tube can be reused for plasmid DNA elution in Step 13.

13. Fit the dried DNA Midi Column onto a clean collection tube, load 0.4 mL Elution Buffer directly onto the center of the silica membrane, incubate for 2 min, then centrifuge at 5,000 rpm for 2 min.

This single elution recovers 60–75% of bound DNA. For maximum DNA concentration, skip the second elution in Step 14 or collect the two eluates separately.

14. Add an additional 0.2 mL Elution Buffer to the membrane center, incubate for 2 min, then centrifuge at 5,000 rpm for 2 min. Discard the column and store purified plasmid at −20 °C.

The silica membrane retains a small volume of liquid, and low centrifugation speed results in ~0.1 mL eluate loss. A second elution efficiently recovers residual bound plasmid DNA. For low-copy vectors, perform the first elution with 0.4 mL buffer and the second with 0.2 mL fresh buffer to obtain a total eluate volume of ~0.5 mL.