Plasmid DNA Isolation protocol, principle, steps… 16th February 2022 – Tags: , , ,

Isolation of plasmid DNA or “plasmid dna isolation” from micro organism is a critical approach in molecular biology and is an crucial step in lots of processes which includes cloning, DNA sequencing, transfection and gene therapy. These processes require the isolation of high purity plasmid DNA.
The purified plasmid DNA can be used immediately in all molecular biology procedures such as restriction enzyme digestion, cloning, PCR, transfection, in vitro translation, blotting and sequencing.

Steps for plasmid DNA extraction

  • Growing the bacterial samples

First, bacterial cells must be cultured in varying amounts of growth medium. Normally, an antibiotic is added to the growth medium, and the plasmid DNA will transmit the antibiotic resistance to the bacteria. You can remove the cells from the growth medium and discard the supernatant by centrifugation. At the cease of this step, the cells becomes mobileular pellets.

  • Resuspend the pelleted cells in the buffer solution.

Next, resuspend the cells in an isotonic solution containing Tris, EDTA (to destabilise the cell wall and prevent plasmid damage), glucose (to prevent cells from bursting) and RNase A (to degrade cellular RNA during cell lysis).

  • Lysis of the cells

Next, an alkaline solution containing sodium hydroxide and sodium dodecyl sulphate (SDS) shall be added to facilitate cell lysis and denaturation of genomic and plasmid DNA together with all proteins in the solution. The highly alkaline solution composed of NaOH and SDS ruptures cell membranes and converts double-stranded DNAs (dsDNA) to single-stranded DNAs (ssDNA).

  • Neutralising the solution with potassium acetate

A potassium acetate solution neutralises the sample and separates plasmid DNA from genomic DNA (gDNA). The smaller plasmid DNA renatures easily, while the larger, more complicated genomic DNA precipitates out of solution.

After centrifugation, the genomic DNA and precipitated proteins form a pellet, while the plasmid DNA remains soluble. The ultimate plasmid DNA withinside the supernatant may be brought on with ethanol or purified the usage of spin-clear out out era or a phenol-chloroform mixture.

  • Precipitate the plasmid DNA with ethanol precipitation.

Finally, you must isolate the plasmid DNA by a process known as ethanol precipitation. Once precipitated, you should rinse the precipitate (the plasmid DNA) in cold 70% ethanol and allow it to dry for about 10 minutes for the alcohol to evaporate. You should also resuspend the DNA pellet in a buffer solution containing Tris, EDTA and RNases to clean up any remaining RNA in the solution.

Tips to keep in mind for plasmid isolation

Plasmid isolation can be challenging, so keep the following tips in mind when extracting plasmid DNA:

Perform cell lysis quickly to avoid irreversible plasmid denaturation.
Resuspension and lysis buffers should be mixed well, so be sure not to combine them vigorously to avoid breaking the DNA into smaller fragments. If they are small enough, broken gDNA can rejoin and remain in solution.
Wear gloves and suitable eye safety while operating with harsh chemical compounds together with NaOH and SDS.

 

Principle

The purification of plasmid DNA from bacterial DNA is based on the differential denaturation of chromosomal and plasmid DNA by alkaline lysis to separate the two. The primary steps of plasmid isolation are disruption of the mobileular shape to create a lysate, separation of the plasmid from the chromosomal DNA, mobileular particles and different insoluble material. Bacteria are lysed or processed with a lysis buffer containing sodium dodecyl sulphate (SDS) and sodium hydroxide.

During this step, maximum of the cells are disrupted, chromosomal and plasmid DNA is denatured and the ensuing lysate is wiped clean through centrifugation, filtration or magnetic stripping. Subsequent neutralisation with potassium acetate lets in most effective the covalently locked plasmid DNA to rejoin and continue to be solubilised. Most of the chromosomal DNA and proteins precipitate in a complicated fashioned with potassium and SDS, that is eliminated through centrifugation.

Which solution is used in plasmid DNA isolation?

This solution contains sodium hydroxide and SDS (sodium dodecyl sulfate). Sodium hydroxide denatures plasmid and chromosomal DNA into single strands.