Isolation of DNA from plant source

The first isolation of DNA was done in 1869 by Friedrich Miescher.

Principle:-
The isolation of DNA usually begins with the lysis of cells or tissues inorder to destroy the protein structures and allows the release of nucleic acids from the nucleus.
The DNA extraction process frees DNA from the cell and then separates it from cellular fluid and proteins so you are left with pure DNA. The three basic steps of DNA extraction are ,
1) lysis
2)precipitation
3) purificatio
CTAB-based protocols tend to work very well,
The ability to extract DNA is of primary importance to studying the genetic causes of disease and for the development of diagnostics and drugs. It is also essential for carrying out forensic science, sequencing genomes, detecting bacteria and viruses in the environment and for determining paternity.

*Protocol:-
1.weight out of 0.4 g of plant sample
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2. Crush the sample in 4000ul CTAB buffer
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3.Immediately transfer the sample in 1.5 ml centrifuge tube
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4. Keep the tubes for incubation for 45-60 min and 72•C. (For protein precipitation)
               
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5. Centrifuge the tubes at 15000 rpm for 15min
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6. Transfer the supernatant to a fresh tubes and add equal volume(24:1) of Chloroform: isoamyl alcohol.
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7. Shake the tubes gently to mix it properly.
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8. Centrifuge the tubes at 15000 rpm for 10 min.
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9. Take the aqueous phase of tubes and transfer it in new tubes.
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10. Add 2/3rd volume of ice chilled isopropanol and keep it for incubation at -20C for overnight.
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11. On Next day remove the tubes from deep freeze and allow it to settle for few minutes.
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12. Centrifuge The tubes at 10000 rpm for 10 minutes.
              
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13. Discard the supernatant and collect the whitish transparent pellet.
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14. Add 100-150 ul of 70% ethanol and centrifuge the tubes at 5000rpm for 5 min.
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15. Collect and air dry the supernatant.
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16. On the basis of quantity of pellet add 50-100ul of TE buffer.
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Note : always maintain the pH of all buffers

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