Isolation Total genomic DNA from Peripheral Blood Leukocytes

Wednesday, June 23, 2004

Description
Following is a modification of Wyman and White PNAS 77:6754-6758 (1980). I've found it a very quick and useful yeilding about 300 ug DNA with a 260/280 ratio of 1.65 - 1.8.

Reference: "Frank, M. B. Isolation of Genomic DNA. In: Frank, M. B. ed. Molecular Biology Protocols. 1997. Oklahoma City. Revision Date: October 2, 1997."

Procedure
1. Start with 10-50 ml peripheral blood in anticoagulant, EDTA is preferred. Centrifuge to pellet the cells at 300xg (1200 rpm in Beckman TJ-6 centrifuge) for 10 minutes at room temperature. Remove and freeze the plasma (if needed). Transfer buffy coat cells to another tube. Resuspend the remaining buffy coat cells and red blood cells in PBS with 0.1% EDTA (1:186 x dilution of 0.5 M EDTA stock) and respin. Add the resulting buffy coat cells to those initially collected, and spin once or twice to reduce red blood cells by transferring leukocytes to a new tube. A final pellet containing about equal volumes of white blood cells and red blood cells is fine.

2. Transfer buffy coat cells and incubate them for 3 hours while gently rotating in 10-20 ml of a pre-heated 55C lysis solution (1 mM EDTA, 10 mM Tris-HCl, pH 7.8, 10 mM NaCl, 1% SDS and 100g/ml proteinase-K).

3. Extract 3-4 times with a phenol/chloroform solution, discarding the lower layer each time. Ether or chloroform extract. You should not see hemoglobin after the final extraction.

4. Ethanol precipitate. Resuspend in 1-2 ml of TE and treat with RNAse A @ 20 g/ml for 1 hour, 37C.

5. Extract 3 times with phenol and the ether. Ethanol precipitate and resuspend in 0.5 ml of TE. A typical yield is 200-400 g of DNA with a 260/280 ratio of 1.6 - 1.8.

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Isolation Total genomic DNA from Peripheral Blood Leukocytes