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Basic Techniques in rDNA Technology

Basic Techniques in rDNA Technology. Dr Imran. Isolation/purification of DNA. Most of the techniques in rDNA technologies will require at least three types of DNA DNA can be purified from several sources

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Basic Techniques in rDNA Technology

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  1. Basic Techniques in rDNA Technology Dr Imran

  2. Isolation/purification of DNA • Most of the techniques in rDNA technologies will require at least three types of DNA • DNA can be purified from several sources e.g animal, plant tissues, bacteria, yeast any other source virus, earth crust, crime scene swabs etc. • Several protocols are available for different tissues on, How to isolate DNA from a particular tissue/organisms

  3. Total Cell DNA /Genomic DNA isolation 1. A culture of bacteria is grown and then harvested. 2.The cells are broken open to release their contents. 3. This cell extract is treated to remove all components except the DNA. 4. The resulting DNA solution is concentrated.

  4. Two types of growth media • Complex (undefined) • Defined

  5. Growing cells • Optimum temperature 37 C • Optimum aeration 150-200 rpm • Optimum nutrient M9 or LB

  6. Calibration curve

  7. Preparation of cell extract • By physical means: Sonication, Pressure, beads etc • By chemical means Lysozyme + EDTA + SDS

  8. Purification of DNA from cell extract • Degradation of impurities Phenol, Chloroform, Protenase K, RNAase • Ion exchange

  9. Ion exchange • DNA and RNA is ………. Negatively or positively charged • Resin with opposite charge • Pour Extract on resin and let it pass • Apply salt solution and elute different contaminating components (proteins and RNA)

  10. Ion exchange

  11. Concentrating DNA

  12. DNA extraction from plant tissue • Cetyltrimethylammonium bromide (CTAB) • NaCl • EtOH

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