Explain the procedure of nucleic acid isolation and purification?

Here is a method -





DNA extraction procedure:





1.Treat 25 mg of tissue with Proteinase K by adding 20 ml of stock PK solution (20 mg/ml) and 180 ul water.


Function: proteinase destroys protein.





2.Let sit at 55 degrees for at least 1 hour.


Function: This temperature is optimal for enzyme reaction; if too high will degrade DNAase.





3.Add 1/10 volume of Kac (potassium acetate) (5 M; pH 5.6) (in this example, 20 ul but remember the 1/10 proportion as a general rule). . Mix well and place on ice for 10 minutes.


Function: precipitates membrane, C, H, O, lipids, proteins, and monomers.





4.Add 200 ml of phenol saturated with 10mM Tris. ( IN HOOD)


Function: removes nonpolar proteins, lipid residue.





5.Cap tube: mix well by inverting tube.





6.Spin 5 minutes in microfuge.





Function: separates aqueous (top) and phenol (bottom) layers


7.Remove upper aqueous layer (CONTAINS DNA) to a new tube.





8.Add 200 ul of chloroform to aqueous layer (IN HOOD).


Function: removes excess phenol from DNA.





9.Cap tub; mix well by inverting.


Function: sets an emulsion; biphasic solution.





10.Remove upper aqueous layer (CONTAINS DNA) to a new tube.





11.Add 500 ul of EtOH and cap tube (again, 500 in this example; as a proportion use twice the total amount).


Function: precipitates DNA; DNA is insoluble in ethanol.





12.Cap tube; mix well by inverting tube several times.





13.Spin for 5 minutes in microfuge. 13000 rpm. (10 min. at 6000.)


Function: collects DNA in bottom of the tube.





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