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, transfer supernatant using a pipet so that particulates are excluded. If the DNA yield is expected to be low (<20 g), add a DNA carrier such as glycogen (10 l of 20 mg/ml glycogen per 6 ml isopro p a n o l ) .
2. Mix the sample by inverting gently 50 times and keep tube at room temperature for at least 5 min.
3.Centrifuge at 2,000 x g for 10 min. The DNA may or may not be visible as a small white pellet, depending on yield.
4. Pour off the supernatant and drain tube briefly 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 pellet to ensure it stays in the tube.
6. I n v e rt and drain the tube on clean absorbent paper and allow to air- d ry for 15 min.
1. Add 100 l of DNA hydration solution (100 l will give a concentration of 200 g/ml if the yield is 20 g DNA).
2. Allow DNA to rehydrate overnight at room temperature . A l t e rn a t i v e l y, heat at 65C for 1 hr. Tap tube periodically to aid in dispersing the DNA.
3. Vo rtex briefly and pulse-spin before use. Store DNA at 28 C .
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