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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.
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.