Protocol Series: Plasmid DNA Isolation from Escherichia coli by Alkaline Lysis Method
The protocol below describes the process for isolation of plasmid DNA by alkaline lysis method. The protocol has become one of the standard protocols in the molecular biology, hence not much modification has been done from the original description present in Molecular Cloning Book by Sambrook and Russel.
- 1.5 ml of overnight grown culture was taken into an Eppendorf tube.
- The cells were then centrifuged at 12,000 rpm for 2 minutes at 4 oC.
- After harvesting, the supernatant was discarded and the pellets were resuspended in 100 µl of Solution I. [Note: Resuspend thoroughly such that no clumps remain]
- Then, 200 µl of Solution II was added and the tubes were mixed by inversion. [Note: Look for the mucoid thread when opening the lid slowly]
- 150 µl of Solution III was added to the mixture and mixed by rotating the tubes.
- The tubes were then incubated on ice for 5 minutes.
- Following this, the tubes were centrifuged at 12,000 rpm for 5 minutes.
- Then, supernatant was taken and transferred to the new tube.
- Equal volume of isopropanol or twice volume of ethanol was added to the tubes and the tubes were vortexed. [Note: Vortex thoroughly for better precipitation]
- Following vortexing, the tubes were centrifuged at 12,000 rpm for 10 minutes.
- The supernatant was discarded and the pellet was washed with 70 % ethanol.
- The tubes were centrifuged at 12,000 rpm for 5 minutes and the supernatant was discarded.
- Then, the tubes were left for drying after which the pellet was dissolved in TE buffer. [Note: Failure to dry the tubes can result in flotation of DNA sample during Agarose gel electrophoresis as well as inhibition of enzymes during further downstreaming processes]
Thus isolated plasmid has been successfully utilized for PCR as well as restriction digestion.
The Alkaline lysis method works on the principle that the plasmid DNA being small can easily renature back while due to its large size, the genomic DNA remains denatured and precipitates out of the solution along with other cellular debris.
Solution I aka Re-suspension buffer has Tris – Cl which acts as a buffer, EDTa which acts as a chelating agent that prevents activation of DNases present in the cell and helps in the protection of the DNA while the glucose helps in disrupting the cell – cell contact, hence helping in the suspension of the Cells.
Solution II aka Lysis Buffer contains a detergenet Sodium Dodecyl Sulfate and Sodium Hydroxide, which acts as a denaturing agent, hence the name of the protocol alkaline lysis, as the cells get ruptured by SDS in presence of NaOH.
Solution III has potassium acetate and glacial acetic acid, which acts a neutralizing agent, hence the name Neutralization buffer. Once the buffer has been added, neutralization occurs which causes renaturation of plasmid DNA due to small size. However, genomic DNA along with majority of cellular debris get precipitated out that can later be separated by centrifugation.
The DNA is washed at the final stage with 70 % ethanol because it is the concentration where most of the DNA remains undissolved while the salts present gets dissolved.
Composition of Solutions:
25 mM Tris pH 8.0
10 mM EDTA pH 8.0
50 mM Glucose
1 % SDS
0.2 N NaOH
5 M Potassium Acetate 60 ml
Glacial acetic acid 11.5 ml
dH2O 28.5 ml
Sambrook J and Russel DW (2001) Molecular Cloning A laboratory Manual (Third Edition): Protocol 1 Preparation of plasmid DNA by alkaline lysis method with SDS: Mini Preparation. Cold Spring Harbor Laboratory Press. 1.32 – 1.34