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DNA extraction from blood

DNA extraction from blood. objective. Isolating DNA molecules from white blood cells and visualizing them directly. Protocol. First: cellular membrane and nucleic membrane must be destroyed lysis buffer

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DNA extraction from blood

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  1. DNA extraction from blood

  2. objective • Isolating DNA molecules from white blood cells and visualizing them directly.

  3. Protocol • First: cellular membrane and nucleic membrane must be destroyed lysis buffer • Secondly: other components of the cell like proteins must be excluded. Chloroform • Finally: DNA can be collected and directly visualized. Sodium acetate and isopropanol

  4. Experiment 1 procedure Get the beaker and add 45ml of water to it then add 5ml of detergent and then add 1 table spoon of salt mix them all together with the spoon. Add few drops of the blood sample to the above solution. Filter the blood solution by filter paper and wait until all the solution is filtered. Take 10ml of the blood solution that has been filtered by using syringe and put it in a small beaker. Then add to that 10ml of isopropanol. finally, we can see the DNA clearly visible in the supernatant layer at the top.

  5. Experiment 2 procedure • In eppendorf tube add 2.5ml of blood with 7.5ml of lysis buffer, shake well and leave it in ice for 5 min. • Put the sample in the centrifuge at 1200,4c for 10min. • Discard supernatant. Add topellet205ml of lysis buffer and centrifuge again at same conditions. • Discard supernatant. Add 1.25ml SE buffer to pellet, centrifuge again at same conditions. • Discard supernatant. Add 1.25ml of SE buffer and 60Ml SDS. Shake gently for 7min. • Add 1.25ml SE buffer and 205ml phenol shake well for 5min then centrifuge 3000,10c for 10min

  6. Transfer supernatant to new tube. Add 2.25ml phenol/chloroform/isoamy alcohol. Shake well for 5min then centrifuge 3000,10c for 10min. • Transfer supernatant to new tube. Add 2.5ml phenol/chloroform/isoamy alcohol. Shake well for 5min then centrifuge 3000,10c for 10min. • Transfer supernatant to new tube. Add 75Ml sodium acetate and 2.5ml isopropanol. Observe DNA fibres visually.

  7. homework • 1-Why do we add detergent as a part of the solution of the DNA blood extraction procedure?? • 2- why do we add table salt as a part of the solution of the DNA blood extraction procedure?? • 3- why do we filter the blood solution in this experiment? • 4- why do we add isopropanol to the filtered DNA??

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