1. Add 1 ml body fluid (e.g., cerebrospinal fluid, plasma, saliva, serum, sputum, synovial fluid, urine, whole blood, milk) to a sterile 15 ml centrifuge tube containing 5 ml cell lysis solution. Pipet up and down several times to mix thoroughly. Note: if the sample has a high protein content, 0.5 ml body fluid may be added to 5.5 ml cell lysis solution.
2. Heat to 65C for 15 min to complete lysis. Alternatively, for maximum yield, add 30 l of proteinase K (20 mg/ml) and incubate lysate at 55C for 1 hr to overnight.
RNase Treatment (Optional)
1. Add 30 l RNase A solution to the cell lysate.
2. Mix the sample by inverting the tube 25 times and incubate at 37C for 1560 min.
1. Cool sample to room temperature.
2. Add 2 ml protein precipitation solution to the lysate.
3. Vortex sample at high speed for 20 sec to mix the protein precipitation solution uniformly with the lysate.
4. Place sample into an ice bath for 515 min.
5. Centrifuge at 2,000 x g for 10 min. The precipitated proteins should form a tight pellet. Note: if body fluid has a high lipid content, particulates may stay near top of tube; see alternative transfer method in step 1 below.
1. Pour the supernatant containing the DNA (leaving behind the p recipitated protein pellet) into a clean 15 ml centrifuge tube containing 6 ml 100% isopropanol (2-propanol). Altern a t i v e l y, if p a rticulates are present,