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 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

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.

Two types of growth media Defined o Complex (undefined)

Growing cells Optimum temperature 37 C Optimum aeration rpm Optimum nutrient M9 or LB

Calibration curve

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

Purification of DNA from cell extract Degradation of impurities Phenol, Chloroform, Protenase K, RNAase 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)

Ion exchange

Concentrating DNA

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