| 1 |
Centrifuge 10 mL of raw milk at 7000 g for 10 minutes (4°C), remove the milk fat and most of the supernatant from above somatic cells and milk proteins pellet, and transfer the pellet with the remainder of the supernatant to a 2-mL sterile tube. Centrifuge the mixture at 5000 × g for 3 minutes (4°C) and remove the supernatant. |
| 2 |
Wash the pellet with 1 mL of buffer (15 mM Tris-HCl (pH 7.4–7.6), 25 mM NaCl, 5 mM MgCl2,15 mM Na2HPO4, 2.5 mM EDTA, 1% sucrose). Centrifuge this mixture at 5000 × g for 3 minutes (4°C), remove the supernatant, and repeat this step until clear supernatant is obtained. |
| 3 |
Add 1 mL of lysis buffer (pH = 8.8; 6% SDS, 3 mM MgCl2,15 mM Tris-HCl, 0.5% DMSO, 6% acetone) to the pellet and incubate this mixture at 65°C for approximately 20–30 minutes. After this time, the strings of DNA clumps will be visible in the liquid. |
| 4 |
Attach the strings of DNA clumps to the wall of a new sterile 1.5-mL tube by pipette. Then, discard leftover supernatant that has dropped to the bottom and wash DNA twice with 100 µL of 70% ethanol. Centrifuge the mixture at 10000 × g for 1 minute at room temperature and discard the supernatant. |
| 5 |
Dissolve the DNA pellet in 50–100 µL of deionized water or TE buffer (pH 8.0, 10 mM Tris,1 mM EDTA) |
| Step |
Procedure 2 |
| 1–2 |
The same as in Procedure 1. |
| 3 |
Add 1 mL of lysis buffer (pH = 8.8; 6% SDS, 3 mM MgCl2,15 mM Tris-HCl, 0,5% DMSO, 6% acetone) to the pellet and incubate this mixture at 65°C for approximately 60–90 minutes. |
| 4 |
Cool down the mixture to room temperature and add 450 µL of protein precipitation buffer (2.35 M NH4Cl, 1.15 M NaCl, 38% ethanol pH 5.0), vortex, and then centrifuge the mixture at 16000 × g for 8 minutes (10°C). |
| 5 |
Transfer the supernatant to a new tube and add 600 µL of 100% isopropanol. Centrifuge the mixture at 10000 × g for 8 minutes and remove the supernatant. |
| 6 |
Wash the DNA pellet twice with 70% ethanol and air dry. |
| 7 |
Dissolve the DNA pellet in 50–100 µL of deionized water or TE buffer (pH 8.0, 10 mM Tris, 1 mM EDTA). |
