DNA Purification by Sanjeev Khanal .pptx

DNA Purifiction

1. DNA Purification DNA purification means removing enzymes, buffers, proteins, lipids, RNA, and other compounds and chemicals that can affect downstream applications. DNA purification is essential for reasons : Ensure that you obtain a sufficient amount of genomic and/or plasmid DNA sample. Reduce the number of contaminants that can compromise your research results.

2. Techniques Used In DNA Purification Plasmid and genomic DNA samples can be purified using several techniques.  Some are: Organic extraction Anion exchange chromatography method Silica-column-based method Magnetic beads-based method

3. Techniques Used In DNA Purification Plasmid and genomic DNA samples can be purified using several techniques.  Some are: a. Organic extraction:  One of the most traditional techniques used to purify DNA. It’s also known as the phenol-chloroform extraction procedure. The  steps followed in this DNA extraction method include: Cell lysis, removing cell debris through centrifugation, and obtaining nucleic acids in the supernatant. Denaturation of proteins followed by their precipitation using a 1:1 mixture of phenol and chloroform. Recovery of purified DNA by precipitating it using ethanol or isopropanol. Degradation of RNA using RNase enzyme . Though it’s an easy-to-perform and cheap approach, this technique is time-consuming and involves the use of hazardous organic solvents.

4. b. Anion exchange chromatography method:  This purification technique involves  the binding of the negatively charged DNA phosphate backbone with the positively charged DEAE to precipitate DNA from the solution. This is achieved by using specific salt and pH conditions and washing steps to remove contaminants like proteins and cellular debris. It provides high-quality DNA with high yield for workflows like DNA sequencing and transfection . c. Silica-column-based method:   It involves the use of chaotropic salts, which provide conditions in which the DNA binds to the silica column , and then elutes from the sample provided opposite condition — in low salt environments . d. Magnetic beads-based method:   At a low pH condition, the DNA bind s to positively charged magnetic beads and at high pH, the beads release the molecule.

5. c. Silica-column-based method d. Magnetic beads-based method