How to isolate plasmid and improve its quality

#1 How to isolate plasmid and improve its quality

The method’s basic idea is to selectively denature high molecular weight chromosomal DNA using alkaline denaturation, leaving covalently closed circular DNA double-stranded. After neutralization, plasmid DNA remains in the supernatant, and chromosomal DNA renatures to form an insoluble clot. Using this technique, plasmid DNAs of all sizes have been isolated.

In recombinant DNA research, bacterial plasmid DNA is frequently used as a cloning vector. Gel electrophoresis can be used to isolate and characterize new plasmids based on their size and restriction enzyme pattern.

Prior research has demonstrated that linear DNA but not CCC-DNA may be denaturated within a specific pH range (about 12.0-12.5), and that this characteristic can be applied to purify CCC-DNA (7–10 pH).

We have applied this strategy to create a quick plasmid DNA extraction method. After weakening the cell wall with lysozyme, plasmid-containing cells are completely lysed using sodium dodecyl sulphate (SDS) and NaOH.

A stable alkaline pH value can be achieved without using a meter by carefully adjusting the ratio of cell suspension to NaOH solution; additional pH control can be achieved by introducing glucose as a pH buffer.

chromosomal DNA, which has nevertheless very high molecular weight. In this manner, the majority of the three main contaminating macromolecules co-precipitate and can be eliminated using a benchtop centrifuge in a single centrifugation.  Ethanol precipitation is used to extract plasmid DNA (as well as any remaining low molecular weight RNA) from the supernatant. Gel electrophoresis can be used to examine plasmid DNA either intact in the CCC form or after restriction enzyme digestion.

Reagents-

A stock solution of RNAse A (1mg/ml in 5mM Tris-HCl, pH 8.0) is treated by heating at 100 ⁰C for 10 min.

I. Lysozyme solution- 2mg/ml lysozyme, 50 mM glucose, 10mM EDTA, 25 mM Tris-HCl (pH 8.0). Make fresh every day using stock solutions of additional components and crystalline lysozyme. Store at 0⁰C.

II. Alkaline SDS solution- 0.2 N NaOH, 1% sodium dodecyl sulfate (SDS). Store at Room temperature. Stable for 1 week.

III. High salt solution- 3M sodium acetate (pH 4.8). To prepare, dissolve two moles of sodium acetate in a small amount of water, use glacial acetic acid to get the pH down to 4.8, and then adjust the volume to one litre. Keep it at room temperature.

Method-

Step1: After 18h incubation, 0.5ml of culture is transferred into centrifuge. All steps are carried out at room temperature except some steps. The tube is centrifuge for 15 seconds.

Step 2: The supernatant is carefully removed and cell pellet is thoroughly suspended in 100 µl of solution I.

Role of Solution I: Solution I is lysis buffer contain EDTA, Tris-HCl, glucose and lysozyme.

EDTA is chelating agent. Mg2+ is important for DNase activity (enzyme that degrade DNA) and by binding Mg+, EDTA inhibits DNAse. It prevents plasmid degradation. EDTA removes divalent cations (Mg2+, Ca2+), making the membrane more permeable to lysis solutions like (SDS and NaOH) and lysis solution weakened cell wall allow SDS to disrupt the membrane, releasing plasmid DNA. Thus, EDTA protects plasmid DNA and improves yield by preventing DNAse degradation and assisting bacterial lyses.

Tris-HCl is buffering agent. DNA is pH sensitive and Tris-HCl prevent acidification of the solution.

The ideal pH for plasmid stability is 8.0. Tris-HCl protects DNA from degradation- Acidic conditions can cause DNA hydrolysis.

Step 3: After incubating at 0°C for 30 minutes, 200 ml of solution II is added, and the tube is gently vortexed. The suspension ought to become somewhat viscous and nearly transparent.

Role of Solution II: SDS is strong anionic detergent. SDS disrupt the phospholipid bilayer of the bacterial cell membrane. This release plasmid DNA and chromosomal DNA into the solution. SDS causes denaturation of proteins and membrane components and making them insoluble. Along with NaOH, SDS denature both chromosomal and plasmid DNA.

Step 4: After keeping the tube at 0°C for 5 minutes, 150 µl of solution III is added. The contents of the tube are gently mixed by inversion for a few seconds during which time a clot of DNA forms. The tube is maintained at 0⁰C, for 60 min. to allow most of the protein, high molecular weight RNA and chromosomal DNA to precipitate.

Role of Solution III: Potassium acetate neutralizes alkaline pH (restore physiological pH)- the previous step denatures both chromosomal and plasmid DNA. Potassium acetate (acidic) neutralize NaOH, allowing plasmid DNA to renature, while chromosomes DNA remains insoluble further leads to the formation of white precipitate of chromosomal DNA, SDS and proteins.

Plasmid DNA remains in the solution and making separation easy.

Step 5: Centrifugation for 5 min and supernatant is removed and transferred to second centrifuge tube. One ml of ethanol is added and the tube is held at -20⁰C, for 30 min.

The precipitate is collected by centrifugation for 2 min and the supernatant removed by aspiration. The pellet is dissolved in 100 µl of 0.1 M sodium acetate/ 0.05M Tris-HCl (pH 8) and re-precipitated with 2 volumes of cold ethanol.

After 10 min at -20 ⁰C, the precipitate is again collected by centrifugation and dissolved in 40 µl MilliQ water and then 10 µl of 5 x sample buffer is added. 10-20 µl is applied to an agarose gel for electrophoresis analysis.

Additional steps to enhance the pure plasmid DNA yield-

After step 4, add equal amount of chloroform. Chloroform denature proteins, helping in their separation from nucleic acids. It forms an organic phase (bottom) and aqueous phase (top)- where plasmid DNA remains. Centrifuge and collect upper layer. Chloroform remove the unwanted protein. It helps extract residual detergent (like SDS) from the lysate. This improves the quality and purity of plasmid DNA.

Now add isopropanol and centrifuge. Isopropanol is used in DNA precipitation and help in concentrating and recovering plasmid DNA from the aqueous solution.

After centrifugation, plasmid DNA forms a pellet at the tube bottom. The supernatant is discarded and the pellet is washed (often 70% ethanol) to removes impurities.

No need to go for step 5.

Add RNase in solution I to remove RNA or can be added after purification.

Reference:

Birnboim, H. Chaim, and Janine Doly. “A rapid alkaline extraction procedure for screening recombinant plasmid DNA.” Nucleic acids research 7.6 (1979): 1513-1523.