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Genomic DNA Extraction Protocol

    Genomic DNA extraction protocol post explains DNA isolation protocol principle, requirements, and procedure.

    Table of Contents

    DNA from cells is required for many biotechnology and research applications, such as molecular cloning. To remove and purify DNA from cells, researchers use various methods for total DNA extraction. While the specifics of different protocols may vary, some general concepts underlie the process of DNA extraction.

    Principle

    Good quality DNA is a prerequisite for all experiments of DNA manipulation. All plant DNA extraction protocols comprise of the basic steps of disruption of the cell wall, cell membrane and nuclear membrane to release the DNA into solution followed by precipitation of DNA while ensuring removal of the contaminating biomolecules such as the proteins, polysaccharides, lipids, phenols and other secondary metabolites.

    Requirements

    Materials

    1. Centrifuge

    2. Deep Freezer

    3. Incubator

    4. Refrigerator

    5. UV-spectrophotometer

    Chemicals

    1. Chloroform

    2. EDTA

    3. Ethanol

    4. Isoamyl Alcohol

    5. Phenol

    6. Proteinase

    7. Ribonuclease A

    8. Sodium Acetate

    9. Sodium Lauryl Sarcosiante

    10. Tris-HCl

    Reagents

    1. Lysis Buffer: Lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5% sodium lauryl sarcosiante, 0.5 mg/ml proteinase K).

    2. RNase A (10mg / ml): Dissolve RNase A in 10mM Tris-Cl, pH 7.5, 15 mM NaCl. Heat at 1000C for 15 min. Cool to room temperature. Store as aliquots at -20°C.

    Procedure

    1. To extract DNA from cells of interest, cells are lysed with 100 –200 ml of lysis buffer [50 mM Tris-HCl (pH 8.0), containing 10 mM EDTA, 0.5% sodium lauryl sarcosinate and 0.5 mg/ml proteinase K]. Incubate for 1 h at 50⁰C.

    2. Add Ribonuclease A (10 ml, 0.5 mg/ml) and incubate for an additional 1 hr at 50⁰C. Add 1ml of phenol, shake well for 5 to 10 min and then centrifuge at 3000 rpm for 5 min at 4⁰C.

    3. Transfer the supernatant to a new microcentrifuges tube with 500 ml of phenol and 500 ml of chloroform/isoamyl alcohol (24:1), shake it well for 5 to 10 min and centrifuge at 3000 rpm for 5 min at 4⁰C.

    4. After centrifugation, transfer the supernatant to a new tube and add 25–50 ml of 3 M sodium acetate (pH 5.2) and 1 ml of ethanol shake gently till the DNA to precipitate.

    5. Then place it under -8⁰C for 20 minutes and centrifuge at 12000 rpm for 20 min to recover the DNA. Rinse the pellet with 1 ml of 70% ethanol and spin for 10min. discard the supernatant and air dry the pellet at room temperature.

    6. Dissolve the DNA in 0.5–1.0 ml of Millipore water to determine the concentration and purity of DNA by absorbance at 260/280 nm in a UV-spectrophotometer.

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