In part (1) of this unit, e. coli will be increased by overnight culture in liquid medium. This is a preparatory step for part (2), where the Plasmid DNA will be isolated using the Alkaline Lysis Method.
The alkaline lysis method uses a high pH (12.1 - 12.5) to denature large sequences of DNA. This allows the selective precipitation of bacterial genomic DNA leaving the smaller plasmid DNA in solution. Other components in the alkaline lysis method remove additional contaminating macromolecules resulting in a relatively clean plasmid DNA sample.
The small-scale plasmid DNA purification performed in this lab is often referred to as a miniprep because it will yield approximately 5-10 μg of plasmid DNA. Plasmid DNA isolated using the alkaline lysis method is pure enough to use as the substrate in an enzyme digestion, for the creation of molecular probes, and DNA libraries just to name a few.
Stages of Alkaline Lysis Method:
a. Resuspension of Cells: Cells are resuspended in Alkaline Lysis Solution I (25 mM Tris pH 8, 50 mM glucose, and 10 mM EDTA, 5 μg/mL of DNase-free RNase). It is very important that the cells are completely resuspended and that there are no clumps left to ensure complete lysis in the next step.
- Tris buffers the solution, allowing the cells to be resuspended in an isotonic solution.
- Glucose maintains the osmolarity.
- EDTA (ethylenediaminetetraacetic acid) is a chelating agent that forms strong complexes with divalent metal cations (primarily Ca2+ and Mg2+). This inactivates DNases present in the cells and destabilizes the cell membrane.
- RNase is added to remove RNA molecules.
b. Cell lysis: Lysis is achieved by the addition of Solution II (1% SDS, 200mM NaOH).
- Sodium dodecyl (lauryl) sulfate (SDS) is a detergent that dissolves the components of cell membranes and denatures cytoplasmic proteins.
- Sodium hydroxide (NaOH) helps break up the cell wall thereby freeing cellular contents into the solution and NaOH denatures DNA. As a result, cellular DNA becomes linearized and denatured (single-stranded) whereas plasmid DNA, being much smaller and supercoiled, remains circular. Being gentle and quick during this step is important as too harsh or too long will affect the plasmid DNA as well.
c. Neutralization of the reaction and clearing of the lysate: Neutralization of the reaction is accomplished with Solution III (3.0M KOAc pH 5.5). The neutral pH allows the plasmid DNA to renature properly (i.e. become double stranded again).
- The potassium acetate (KOAc) drops the pH dramatically, making it neutral. This causes the genomic DNA and proteins to aggregate and form an insoluble complex that precipitates due to the high concentration of salt.
- KOAc interacts with SDS forming potassium dodecyl sulfate, which is insoluble and precipitates along with the cellular proteins associated with it.
Now in the solution: The genomic DNA and proteins are precipitated, while the plasmid DNA is renatured in the solution.
d. Precipitation and de-salting: A specific concentration of alcohol (ethanol or isopropanol), along with a salt (e.g. ammonium acetate, lithium chloride, or sodium acetate) that masks the charges on the DNA will precipitate DNA. In this case ethanol and KOAc, which is already in the solution, are used to precipitate the plasmid DNA.
e. A further wash with 70% ethanol is performed to remove residual salt from the already precipitated plasmid DNA.