Plasmid Isolation by Alkaline Lysis Method

Alkaline lysis method of plasmid isolation was originally developed by Birnboim & Doly (1979). In this procedure, bacteria harbouring the desired plasmid are harvested from the culture medium. Suspension of harvested bacterial cells are made in an isotonic solution which is subsequently subjected to lysis by an alkaline solution containing a detergent (SDS) and NaOH. While detergent serves to lyse cells and denature proteins, alkaline conditions denature genomic DNA, plasmid DNA as well as proteins. This mixture in the subsequent step is neutralized by potassium acetate (pH 5.2). Neutralization results in renaturation of plasmid and genomic DNA. Since plasmid DNA is covalently closed, it reanneals properly and remains in solution in soluble form while genomic DNA reanneals random, resulting in the formation of precipitate. High speed centrifugation separates the precipitate from the aqueous phase (supernatant). Plasmid from the supernatant can be recovered by precipitation using isopropanol or ethanol.

Alkaline lysis method of plasmid isolation from E. coli can have the following steps

  1. Harvesting of bacteria from culture
  2. Resuspension of harvested bacteria in resuspension buffer
  3. Lysis in presence of alkaline condition
  4. Neutralization of lysate to precipitate genomic DNA
  5. Clearing of lysate
  6. Recovery of plasmid DNA from cleared lysate
  7. Washing of plasmid DNA to remove impurities
  8. Storage of plasmid DNA

Step 1: Harvesting of bacteria from culture
Generally bacterial cells containing the plasmid are grown in a liquid medium. Therefore, it is essential to separate bacterial cells from the culture medium. Almost all protocols use centrifugation to separate the bacterial cells. Centrifugation speed should be optimized in such a way that it results in accumulation of all the bacterial cells in a form of pellet and at the same time resulting pellet should be loose enough to be resuspended easily in the resuspension buffer. Centrifugation at high speed and more time can result in a hard pellet which would be difficult to resuspend. Clumps of bacteria in the resuspension buffer leads to incomplete lysis, resulting in low yield of plasmid. Separating bacterial cells from the rest of the culture medium is essential as bacterial culture medium may contain inhibitors which can inhibit the enzyme activity (e.g., restriction enzymes). To avoid such problems, a wash with Tris.EDTA (pH 8.0) or resuspension buffer is recommended.

Step 2: Suspension of bacterial pellet in resuspension buffer
The bacterial pellet isolated from step first is resuspended in a resuspension buffer. Resuspension buffer contains Glucose, EDTA and Tris.Cl. Glucose is required to make the solution isotonic. EDTA chelates the divalent cations which are released upon bacterial lysis. Divalent cations are required for many enzymatic reactions. EDTA action results in inactivation of many enzymes which may harm plasmid DNA. Tris.Cl acts as a buffering agent. Now researchers prefer to supplement the resuspension buffer with RNase A. RNase A is a very stable enzyme and is active under very stringent conditions including high alkaline condition, presence of detergent and chelating agent (EDTA). Addition of RNase A in the resuspension buffer helps to remove RNA from the plasmid preparation. In the subsequent lysis step, RNase A digests the RNA of the bacteria.

Step 3: Lysis of bacteria
In this step, bacterial suspension is treated with lysis solution which is nothing but the alkaline solution of sodium dodecyl sulfate (SDS) (NaOH containing SDS solution, pH ≈ 12.5). Alkaline solution of SDS is highly efficient at lysing the cells by solubilizing the phospholipid and denaturing protein components of the cell membrane, thus releasing the cell contents including plasmid DNA and Genomic DNA. High alkaline condition due to NaOH also denatures the plasmid and genomic DNA.

Step 4: Neutralization of lysate
Addition of neutralization solution (acidic potassium acetate) brings the lysate pH back to normal, resulting in precipitation of protein and genomic DNA. Both plasmid and genomic DNA renatures upon addition of neutralization buffer. While plasmid DNA renatures in correct conformation due to its circular and covalent nature, therefore, remains in the solution, genomic DNA precipitates due to random association of both the strands. Sodium dodecyl sulfate (SDS) reacts with potassium acetate and forms insoluble potassium dodecyl sulfate (KDS).

Step 5: Clearing of lysate
The precipitate formed upon addition of the neutralization solution is separated by centrifugation at high speed. Some commercially available methods provide columns which function like a stainer.

Step 6: Recovery of plasmid from cleared lysate
Cleared supernatant contains plasmid. Most protocols use precipitation to recover plasmid from solution. Plasmid DNA can be precipitated by either addition two volumes of absolute alcohol or 0.7 volume of isopropanol to the cleared lysate. Centrifugation at high speed results in collection of plasmid in a form of pellet.

Step 7: Washing of plasmid pellet
The precipitated plasmid DNA (pellet) contains salts which must be removed from the plasmid. For this purpose 70% ethanol wash is given to the pellet.

Step 8: Dissolving and Storage of Plasmid
The plasmid pellet can be dissolved in double distilled water or TE (pH 8.0) and can be stored at -20°C.

REFERENCES:

Birnboim & Doly, 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7(6), 1513-23. PMID-388356; Full Text Link: academic.oup, PMC342324

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