- Alkaline lysis method of plasmid isolation was originally developed by Brinboim and Doly (1979). In this procedure, bacteria containing the desired plasmid are harvested from culture medium. Suspension of bacteria is made in 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 condition denature genomic DNA, plasmid DNA as well as proteins. This mixture in 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. Precipitate is separated by high speed centrifugation. Plasmid from the supernatant can be recovered by precipitation using isopropanol or ethanol.
A typical isolation procedure by alkaline lysis have the following steps.
1. Harvesting of baceria from culture
2. Suspension of bacterial pellet in resuspension buffer
3. Lysis of bacteria
4. Neutralization of lysate
5. Clearing of lysate
6. Recovery of plasmid from cleared lysate
7. Washing of plasmid DNA pellet
8. Storage of Plasmid DNA
1. Harvesting of baceria from culture :
Generally bacterial cells containing the plasmid are grown in a liquid media. Therefore it is essential to separete the bacterial cells from the culture medium. Almost all protocols use centrigugation to separate the bacterial cells. Centrifugation speed is 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 resuspention buffer. Clumps of bacteria in 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.
2. Suspension of bacterial pellet in resuspension buffer:
Bacterial pellet isolated from step first is resuspended in resuspension buffer. Resuspension buffer contains Glucose, EDTA and Tris.Cl. Glucose is required to make the solution isotonic. EDTA chelate 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 resuspension buffer with RNase A. RNase A is a very stable enzyme and is active under very stringent condition including high alkaline condition, presence of detergent and chelating agent (EDTA). Addition of RNase A in resuspension buffer helps to remove RNA from the plasmid preparation. In the subsequent lysis step, RNase A digests the RNA of the bacteria.
3. Lysis of bacteria:
In this step, bacterial suspension is treated with lysis solution which contains Sodium dodecyl sulfate (SDS) and NaOH. SDS is a detergent which solubilizes the phospholipid and denatures protein components of the cell membrane, leading to lysis and release of the cell contents. High alkaline condition due to NaOH denatures the plasmid and genomic DNA.
4. Neutralization of lysate:
Addition of neutralization solution (Acidic potassium acetate) bring 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 asociation of both the strands. Sodium dodecyl sulfate (SDS) reacts with potassium acetate and form insoluble potassium dodecyl sulfate (KDS).
5. Clearing of lysate:
Precipitate formed upon addition of neutralization solution is separated by aquous solution by centrifugation at high speed. Some commercially available methods provide columns which function like a stainer.
6. Recovery of plasmid from cleared lysate:
Cleared lysate 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.
7. Washing of plasmid pellet:
Precipitation of plasmid DNA contains salts which need to be removed from the plasmid. For this porpose 70% ethanol wash is given to the pellet.
8. Storage of Plasmid:
Obtained plasmid pellet can be dissolved in double distilled water or TE (pH 8.0).