# Alkaline Lysis Plasmid Miniprep Protocol > Published on Vicena | [View on web](https://vicena.ai/wiki/alkaline-lysis-plasmid-miniprep-protocol-rdQWhg) --- ## User What is the step-by-step protocol for alkaline lysis plasmid miniprep, including buffer compositions for P1 P2 and P3, centrifugation times, isopropanol precipitation, and expected DNA yield? --- ## Vicena The alkaline lysis method, originally described by [Birnboim and Doly (1979)](https://pubmed.ncbi.nlm.nih.gov/492765/), remains the gold standard for plasmid DNA isolation. The process relies on the differential denaturation and renaturation of plasmid versus chromosomal DNA. ### 1. Buffer Compositions Standard formulations for the three primary buffers are as follows: | Buffer | Name | Composition | Function | | :--- | :--- | :--- | :--- | | **P1** | Resuspension | 50 mM Tris-Cl (pH 8.0), 10 mM EDTA, 100 $\mu$g/mL RNase A | Destabilizes the cell wall (EDTA chelates $\ce{Mg^2+}$) and degrades RNA. | | **P2** | Lysis | 200 mM $\ce{NaOH}$, 1% (w/v) SDS | $\ce{NaOH}$ denatures DNA; SDS solubilizes the membrane and denatures proteins. | | **P3** | Neutralization | 3.0 M Potassium Acetate (pH 5.5) | Neutralizes pH, causing $\ce{KDS}$ and proteins to precipitate, trapping genomic DNA. | * **Preparation Tip:** Buffer P3 is prepared by dissolving 294.5g of potassium acetate in 500mL $\ce{H2O}$, adjusting pH to 5.5 with ~110mL glacial acetic acid, and bringing the volume to 1L. --- ### 2. Step-by-Step Protocol This protocol is optimized for a standard 1.5–5 mL *E. coli* culture. 1. **Harvest Cells:** Centrifuge the bacterial culture at **>10,000 x g for 1–3 minutes**. Discard the supernatant completely. 2. **Resuspension (P1):** Add 250 $\mu$L of **Buffer P1**. Vortex or pipet up and down until the pellet is completely resuspended (no clumps). 3. **Lysis (P2):** Add 250 $\mu$L of **Buffer P2**. Mix by gently inverting the tube 4–6 times. * *Warning:* Do not vortex. Do not exceed **5 minutes** of incubation, as this can lead to irreversible denaturation of the plasmid DNA. 4. **Neutralization (P3):** Add 350 $\mu$L of **Buffer P3**. Mix immediately but gently by inversion 4–6 times. A white, flocculent precipitate (genomic DNA, proteins, and $\ce{KDS}$) will form. 5. **Clarification:** Centrifuge at **maximum speed (≥13,000 x g) for 10 minutes**. A compact white pellet should form at the bottom/side. 6. **Isopropanol Precipitation:** * Carefully transfer the clear supernatant (containing the plasmid) to a fresh tube. * Add **0.6–0.7 volumes** of room-temperature isopropanol (approx. 500–600 $\mu$L). * Mix well by inversion and incubate at room temperature for 2–10 minutes. 7. **DNA Recovery:** Centrifuge at **maximum speed for 15–20 minutes** at 4°C (preferred) or room temperature. The DNA pellet may be small and translucent. 8. **Washing:** Discard the supernatant. Add 1 mL of **70% ethanol** to wash the pellet. Centrifuge at maximum speed for 5 minutes. 9. **Drying & Resuspension:** Remove all ethanol. Air-dry the pellet for 5–10 minutes (do not over-dry, or it will be hard to dissolve). Resuspend in 30–50 $\mu$L of **TE buffer** or nuclease-free water. --- ### 3. Expected Yield and Optimization * **Expected Yield:** For a high-copy plasmid (e.g., pUC vectors) in LB media, a standard 1.5 mL miniprep typically yields **10–20 $\mu$g** of DNA. Low-copy plasmids may yield **<5 $\mu$g**. * **Yield Maximization Tips:** * **Culture Volume:** For low-copy plasmids, use up to 10 mL of culture and [double the volumes](https://www.qiagen.com/us/products/discovery-and-translational-research/lab-essentials/buffers-reagents/plasmid-buffers) of P1, P2, and P3. * **Precipitation:** Using **ice-cold isopropanol** or adding 1/10 volume of 3M Sodium Acetate (pH 5.2) before the alcohol can increase recovery of very small amounts of DNA. * **Incubation:** If yields are low, incubate the isopropanol/DNA mixture at **-20°C for 30 minutes** before the final centrifugation. ---