Here's the science...

DNA Extraction

Materials:
• DNA extraction buffer =100 mM Tris-HCl [pH 8.0], 100 mM sodium EDTA [pH 8.0],
100 mM sodium phosphate buffer [pH 8.0], 1.5 M NaCl)
• Proteinase K (10mg/ml) – degrades DNAases, RNAases, and proteins
• 20% SDS solution
• Chloroform – hydrophobic; stops foaming
• Isopropanol
• 70% ethanol (EtOH)
• Lysozyme – breaks down glycans

1. Filters were cut into small pieces and added to a 15 ml centrifuge tube.
2. 50 µl of proteinase K was added to the tube. Additionally, 0.25 g acid washed PVPP (polyvinyl polypyrrolidone; removes humic acid and esters) and 50 ?l lysozyme (20 mg/ml) were added.
3. Place tube in 37?C incubator for 30 minutes. Mix every 5 minutes if a shaking incubator is not available.
4. Add 0.6 ml 20% SDS solution to each tube.
5. Incubate at 65?C for 2 hours. Gently invert tube end to end every 20 minutes.
6. Centrifuge sample at 6000xg for 10 minutes.
7. Transfer supernatant to new sterile, DNA free tube.
8. Resuspend pellet in 2.25 ml DNA extraction buffer and 0.25 ml 20% SDS solution.
9. Incubate for 10 minutes at 65ºC.
10. Repeat steps 7-8-9 one time for a total of 3 centrifugations and 3 (10 min.) incubations. Pool supernatant from repeated steps in a centrifuge tube and mix. Discard the pellet. Seven ?l of supernatant was recovered.
11. An equal volume of chloroform was added to the supernatant and mixed well.
12. Centrifuge at 6000xg for 10 min. (or max. in clinical centrifuge).
13. Remove the top, aqueous layer being careful not collect emulsion of chloroform and place in DNA-free tube. You should recover close to the original volume.
14. Add 0.6 ml volume isopropanol to supernatant and let DNA precipitate at room temperature for 1 hour (alternatively, the supernatant-isopropanol could be placed in refrigerator over night).
15. Place 1.5 mls in to ~ 10 microcentrifuge tubes. Centrifuge at max. speed in microcentrifuge for 30 min., room temperature. There should be a faint pellet at the bottom that is your DNA. Decant off the supernatant and add about 75 ?l 70% EtOH to each tube.
16. Vortex vigorously to suspend pellet and transfer DNA to a sterile microcentrifuge tube. Add another 75 ?l 70% EtOH to each tube to retrieve residual and add to same tube.
17. Centrifuge in microcentrifuge at 1400xg for 10 min. Remove ethanol. Repeat if necessary.
18. Dry DNA in laminar hood ~ 1 hour or until dry.
19. Resuspend DNA in 50 -100µl sterile distilled (Millipore) H2O. Store at -4ºC.