Mar 23, 2017

Public workspaceIsolation of plasmid DNA from E. coli (Alkaline lysis method)

DOI
dx.doi.org/10.17504/protocols.io.gtbbwin
  • Anna Behle1
  • 1Institute for Synthetic Microbiology, Heinrich-Heine-University, Düsseldorf
  • Axmann Lab
Anna Behle
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.gtbbwin
Protocol Citation: Anna Behle 2017. Isolation of plasmid DNA from E. coli (Alkaline lysis method). protocols.io https://dx.doi.org/10.17504/protocols.io.gtbbwin
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
Created: December 19, 2016
Last Modified: March 28, 2018
Protocol Integer ID: 4675
Abstract
Isolation of plasmid DNA from E. coli using the alkaline lysis method modified from Birnboim et al., 1979. 
This protocol is suitable for fast, cheap recovery of large amounts of plasmid, e.g. for cloning purposes or restriction analysis. However, the purity of plasmid is insufficient for sequencing.
Safety warnings
The purity of the plasmid is insufficient for sequencing and therefore should be cleaned up beforehand using a spin column or chloroform/phenol extraction.
Buffers required
Buffers required
  • Buffer P1:
            50 mM Tris-HCl, pH 8.0; 10 mM EDTA; 100 mg/mL RNase A, (store at 4˚C)
  • Buffer P2:
            0.2 M NaOH; 1 % (w/v) SDS
  • Buffer P3:
            3 M Potassium acetate, pH 5.5
Inoculation
Inoculation
Inoculate 5 mL LB, supplemented with appropriate antibiotics, using a single colony. Grow over night at 37˚C.
Cell harvest
Cell harvest
 Centrifuge culture at maximum speed and RT for 1 min. 
Cell lysis
Cell lysis
Completely remove supernatant. Resuspend in 350 µL buffer P1.
Add 350 µL buffer P2. Gently mix by inverting (do not vortex!). Incubate for up to 5 min at RT. (Note: If incubated for too long, sheared genomic DNA fragments may contaminate the sample.)
Duration00:05:00
Add 400 µL buffer P3. Gently mix by inverting (do not vortex!).
Centrifuge for 10 minutes at RT and maximum speed
Carefully transfer supernatant to a fresh tube, with as little contamination of pelletted material as possible. If necessary, centrifuge the supernatant again (Step 7) to completely remove pellet
Isopropanol precipitation
Isopropanol precipitation
Add 1 volume of isopropanol. Incubate on ice for at least 2 minutes. (Note: Incubation time can be increased to an hour, if required.)
Duration00:02:00
Centrifuge sample for 5 minutes at RT and maximum speed. Carefully remove supernatant without discarding the DNA pellet.
Wash pellet by adding 500 µL of 70 % ethanol (do not resuspend). Centrifuge sample for 5 minutes at RT and maximum speed.
Dry pellet in a thermoblock at 65 ˚C for 10 minutes.
Duration00:10:00
DNA recovery
DNA recovery
Resuspend pellet in 50-100 µL pure water or buffer of choice (e.g. TE buffer).
Optional: Sample can be incubated at 65 ˚C to dissolve DNA more efficiently.