Sep 08, 2025

Public workspaceAlkaline Lysis Plasmid Purification

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvmby9og3p/v1
  • Agustín Sanchez-Temprano1
  • 1CICA - Centro Interdisciplinar de Química e Bioloxía.
Agustín Sanchez-Temprano
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvmby9og3p/v1
Protocol Citation: Agustín Sanchez-Temprano 2025. Alkaline Lysis Plasmid Purification . protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmby9og3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 05, 2025
Last Modified: September 08, 2025
Protocol Integer ID: 226560
Keywords: plasmid dna purification by alkaline lysi, plasmid dna purification, alkaline lysis plasmid purification, plasmid dna, unique structural properties of plasmid dna, quality plasmid suitable for downstream application, quality plasmid, chromosomal dna, dna, alkaline lysi, cloning, such as cloning, molecular biology technique, used molecular biology technique
Abstract
Plasmid DNA purification by alkaline lysis is a widely used molecular biology technique for isolating plasmid DNA from Escherichia coli cultures. This method leverages the unique structural properties of plasmid DNA to separate it effectively from chromosomal DNA and cellular debris, yielding high-quality plasmid suitable for downstream applications such as cloning, sequencing, PCR, and transfection.
Guidelines
  • Use sterile techniques to avoid contamination that may degrade plasmid DNA.
  • Adjust reagent volumes proportionally according to the culture volume (mini, midi, or maxi prep).
  • Mix buffers and cell suspensions gently by inversion rather than vortexing to prevent shearing of genomic DNA.
  • Keep all buffers and samples cold when indicated to maintain plasmid stability and reduce nuclease activity.
  • Always include RNase A in the resuspension buffer to remove RNA contamination.
  • Perform centrifugation steps at recommended speeds and temperatures to ensure efficient separation of plasmid DNA.
  • Validate plasmid DNA quality and concentration post-prep by spectrophotometry or gel electrophoresis.
Materials
Resuspension Buffer
  • 50 mM Tris base
  • 10 mM EDTA
  • 100 µg/ml of RNAse (optional)
  • pH 8.0
Lysis Buffer
  • 200 mM NaOH
  • 1% SDS
Neutralization Buffer
  • 3M Potassium acetate
  • pH 5.0
Elution Buffer
  • 10 mM Tris base
  • 1 mM EDTA
  • pH 8.0
Isopropanol
Ethanol
Sterile Water
Shaking incubator
Laboratory centrifuge

Troubleshooting
Safety warnings
  • Sodium hydroxide (NaOH) and sodium dodecyl sulfate (SDS) are caustic reagents: wear gloves, protective eyewear, and lab coat when handling.
  • Avoid vigorous vortexing or shaking after lysis buffer addition to reduce shearing of chromosomal DNA that contaminates plasmid preps.
  • Do not exceed recommended incubation times during lysis to prevent irreversible denaturation of plasmid DNA.
  • Handle pellets gently during washing and resuspension to avoid loss of plasmid DNA.
  • Dispose of bacterial waste and chemical reagents according to institutional biosafety and chemical safety guidelines.
Before start
  • Prepare all buffers fresh or ensure they are properly stored and equilibrated to appropriate temperatures before starting.
  • Pre-cool centrifuges, tubes, and neutralization buffer to 4°C or on ice as required.
  • Grow E. coli cultures overnight in LB medium with appropriate antibiotics to reach stationary phase (14–16 hours, 37°C, shaking).
  • Confirm culture density to avoid overloading the lysis step, which can reduce purity and yield.
  • Have all materials including pipettes, filter tips, tubes, and reagents ready and labeled for efficient workflow.
Bacterial culture
Inoculate E. coli harboring the plasmid into LB broth with appropriate antibiotic.
  • Mini prep: Amount2-5 mL
  • Midi prep: Amount25-100 mL
  • Maxi prep: Amount250-1000 mL
Incubate DurationOvernight at Shaker150 rpm, 37°C
Incubation
Overnight
Bacterial harvesting
2m
Centrifuge
  • Mini prep: Centrifigation12000 x g, 4°C, 00:01:00
  • Midi prep:Centrifigation10000 x g, 4°C, 00:10:00
  • Maxi prep: Centrifigation10000 x g, 4°C, 00:10:00
2m
Centrifigation
Discard superntatant completely.
Resuspension
Resuspend cell pellet in resuspension buffer:
  • Mini prep: Amount250 µL
  • Midi prep: Amount4 mL
  • Maxi prep: Amount10 mL
Mix gently by pipetting until cells are completely resuspended.
Alkaline lysis
4m
Add lysis buffer to resuspended cells:
  • Mini prep: Amount250 µL
  • Midi prep: Amount4 mL
  • Maxi prep: Amount10 mL
Mix gently by inverting the tube 4-6 times. Avoid vortexing to prevent shearing genomic DNA.
Incubate at TemperatureRoom temperature forDuration00:04:00 . Do not exceed 5 minutes to prevent irreversible denaturation.

4m
Incubation
Critical
Neutralization
10m
Add ice-cold neutralization buffer
  • Mini prep: Amount350 µL
  • Midi prep: Amount6 mL
  • Maxi prep: Amount15 mL
Mix gently by inverting until a thick withe precipitate forms.
Incubate on ice for Duration00:10:00 to enhance precipitate formation.

10m
Incubation
Lysate clearing.
Centrifuge
  • Mini prep: Centrifigation12000 x g, 4°C, 00:10:00
  • Midi prep: Centrifigation10000 x g, 4°C, 00:15:00
  • Maxi prep: Centrifigation10000 x g, 4°C, 00:15:00
Centrifigation
  • Carefully transfer the clear supernatant to a fresh tube without disturbing the pellet.
Precipitation of plasmid DNA
30m
Add ice-cold isopropanol
  • Mini prep: Amount250 µL
  • Midi prep: Amount4 mL
  • Maxi prep: Amount10 mL
Mix by gentle inversion.
Incubate at Temperature-20 °C for Duration00:30:00

30m
Incubation
Centrifuge
  • Mini prep: Centrifigation12000 x g, 4°C, 00:15:00
  • Midi prep: Centrifigation10000 x g, 4°C, 00:15:00
  • Maxi prep: Centrifigation10000 x g, 4°C, 00:15:00
45m
Centrifigation
  • Discard supernatant gently without disturbing pellet.

Wash DNA pellet
5m
Add Amount1 mL of Concentration70 % (v/v) ice-cold ethanol.
Centrifuge
  • Mini prep: Centrifigation12000 x g, 4°C, 00:15:00
  • Midi prep: Centrifigation10000 x g, 4°C, 00:15:00
  • Maxi prep: Centrifigation10000 x g, 4°C, 00:15:00
Centrifigation
  • Remove ethanol carefully and air-dry pellet for Duration00:05:00 . Avoid over-drying.
5m
Critical
Resuspension
5m
Resuspend DNA pellet TE buffer or nuclease-free water.

  • Mini prep: Amount50-100 µL
  • Midi prep: Amount500-2000 µL
  • Maxi prep: Amount5-20 mL
  • Incubate at Temperature37 °C or TemperatureRoom temperature for Duration00:05:00 until fully dissolved.

5m
Incubation
Protocol references
Birnboim, H. C., & Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic acids research, 7(6), 1513–1523. https://doi.org/10.1093/nar/7.6.1513.

Green, M. R., & Sambrook, J. (2018). Preparation of Plasmid DNA by Alkaline Lysis with Sodium Dodecyl Sulfate: Maxipreps. Cold Spring Harbor protocols, 2018(1). https://doi.org/10.1101/pdb.prot093351.