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Genomic DNA Isolation Protocol for 40 l Avian Whole Blood


AquaPure Genomic DNA Isolation Kit
Catalog #732-6340
Expected yield range: 100300 g DNA


Method
Cell Lysis
1. Use fresh blood within 10 sec of venipuncture to avoid clotting. Alternatively, collect whole blood in EDTA to prevent the blood from clotting and reduce DNA degradation. Blood may be either fresh or frozen. Store fresh blood sample at 28C for not more than 5 days to obtain optimal DNA isolation results.

2. Add 40 l whole blood to a 15 ml microfuge tube containing 6 ml cell lysis solution. Quickly pipet up and down 35 times to lyse the cells. Usually no incubation is required; however, if cell clumps are visible after mixing, incubate at 37C until the solution is homogeneous. Samples are stable in cell lysis solution for at least 18 months at room temperature.

RNase Treatment (Optional)
1. Add 30 l RNase A solution to the cell lysate.

2. Mix the sample by capping the tube and inverting it 25 times, then incubate at 37C for 1560 min.

Protein Precipitation
1. Cool sample to room temperature.

2. Add 2 ml protein precipitation polution to the RNase A treated cell lysate.

3. Vortex vigorously at high speed for 20 sec to mix the protein precipitation solution uniformly with the cell lysate.

4. Centrifuge at 2,000 x g for 10 min. The precipitated proteins will form a tight pellet. If the protein pellet is not visible or is not tight, repeat step 3, incubate on ice for 5 min, then repeat step 4.

DNA Precipitation
1. Leaving behind the precipitated protein pellet, pour the supernatant containing the DNA into a 15 ml centrifuge tube containing 6 ml 100% isopropanol (2propanol).

2. Mix the sample by gently inverting the capped tube 50 times.

3. Centrifuge at 2,000 x g for 3 min; the DNA will be visible as a small white pellet.

4. Pour off the supernatant and drain tube on clean absorbent paper. Add 6 ml 70% ethanol and invert the capped tube several times to wash the DNA pellet.

5. Centrifuge at 2,000 x g for 1 min. Carefully pour off the ethanol. Pellet may be loose, so pour slowly and watch to ensure that pellet stays in the tube.

6. Invert and drain the tube on clean absorbent paper and allow to air-dry 15 min.

DNA Hydration
1. Add 500 l DNA hydration solution (500 l will give a concentration of 400 g/ml if the total yield is 200 g DNA).

2. Allow DNA to rehydrate overnight at room temperature. Alternatively, heat at 65C for 1 hr. Tap tube periodically to aid in dispersing the DNA.

3. Store DNA at 28C.


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