Phenol-free method of isolating DNA

1. A phenol-free method of isolating DNA from biological material, comprising:

homogenizing a biological material with a homogenization buffer to form a homogenate, the homogenization buffer comprising a chaotropic guanidine compound and 2-amino-2-(hydroxymethyl)-1-propanediol, hydrochloride (Tris HCl);

extracting proteins and non-DNA organic molecules from the homogenate by mixing a first extraction buffer and a second extraction buffer with the homogenate, the first extraction buffer comprising chloroform and an alcohol and the second extraction buffer comprising a non-ionic protein solubilizer and an alcohol; and

precipitating DNA from the mixture of homogenate, first extraction buffer and second extraction buffer and recovering the DNA by sedimentation.

2. The method of claim 1 , wherein the homogenization buffer comprises guanidine thiocyanate.

3. The method of claim 2 , wherein the homogenization buffer has a pH of about 7.

4. The method of claim 1 , wherein the first extraction buffer comprises chloroform and iso-amyl alcohol.

5. The method of claim 1 , wherein the second extraction buffer comprises an alcohol and octylphenolpoly(ethyleneglycolether).

6. The method of claim 1 , further comprising mixing the biological material with a lysis buffer prior to homogenizing the biological material.

7. The method of claim 6 , the lysis buffer comprising Proteinase K.

8. The method of claim 6 , the lysis buffer comprising Proteinase K, Sodium Dodecyl Sulfate (SDS), Tris-HCl, NaCl, and CaCl 2 .

9. The method of claim 1 , the DNA being precipitated from the mixture of homogenate, first extraction buffer, and second extraction buffer by adding a lower alcohol to the mixture.

10. The method of claim 1 further comprising the step of contacting the recovered DNA with a basic re-suspension solution, the re-suspension solution comprising RNase A.

11. A phenol-free method of isolating DNA from biological material, comprising:

homogenizing a biological material with a homogenization buffer to form a homogenate, the homogenization buffer comprising a guanidine thiocyanate and 2-amino-2-(hydroxymethyl)-1-propanediol, hydrochloride (Tris HCl);

extracting proteins and non-DNA organic molecules from the homogenate by mixing a first extraction buffer and a second extraction buffer with the homogenate, the first extraction buffer comprising chloroform and iso-amyl alcohol and the second extraction buffer comprising octylphenolpoly(ethyleneglycolether); and

precipitating DNA from the mixture of homogenate, first extraction buffer and second extraction buffer by adding a lower alcohol to the mixture and recovering the DNA by sedimentation.

12. The method of claim 11 , wherein the homogenization buffer has a pH of about 7.

13. The method of claim 11 , wherein the second extraction buffer has a pH of about 5.2.

14. The method of claim 11 , further comprising mixing the biological material with a lysis buffer prior to homogenizing the biological material.

15. The method of claim 14 , the lysis buffer comprising Proteinase K, SDS, Tris-HCl, NaCl, and CaCl 2 .

16. The method of claim 11 , further comprising the step of contacting the recovered DNA with a basic re-suspension solution, the re-suspension solution comprising RNase A.

17. A phenol-free method of isolating DNA from biological material comprising the steps of:

homogenizing a biological material with a homogenization buffer to form a homogenate, the homogenization buffer comprising about 4 M guanidine thiocyanate and about 50 mM Tris-HCl at about pH 7.0;

extracting proteins and non-DNA organic molecules from the homogenate by mixing a first extraction buffer and a second extraction buffer with the homogenate, the first extraction buffer comprising about 99% chloroform (v/v) and about 1% iso-amyl alcohol (v/v), the second extraction buffer comprising about 1.0% octylphenolpoly(ethyleneglycolether) solution (v/v), about 0.2% iso-amyl alcohol (v/v), and about 0.3 M sodium acetate (v/v) at about pH 5.2; and

precipitating DNA from the mixture of homogenate, first extraction buffer and second extraction buffer by adding a lower alcohol to the mixture and recovering the DNA by sedimentation.

18. The method of claim 17 further comprising the step of mixing the biological material with a lysis buffer prior to homogenization, the lysis buffer comprising about 100 mM Tris-HCl at about pH 8.0, about 0.5% SDS (w/v), about 100 mM NaCl, and about 2 mM CaCl 2 .

19. The method of claim 18 , wherein the lysis buffer further comprising an amount of Proteinase K effective to bring the final concentration of Proteinase K in the lysis buffer to about 200 μg/mI.

20. The method of claim 17 , further including the step of contacting the recovered DNA with a re-suspension solution, the re-suspension solution comprising about 10 mM Tris-HCl, at about pH 8.5, and about 10 μg/mI of RNase A.