Kit-Free DNA Extraction from Hair (DTT Protocol)

Step 1. Collect hair sample into a 1.5 ml microcentrifuge tube.

Step 2. Add 500 μl digestion buffer.

Step 3. Add 40 μl of 1M DTT.

Step 4. Add 15 μl of 10 mg/ml proteinase K.

Step 5. Vortex briefly.

Step 6. Incubate overnight at 56 °C.

12 hr @ 56 °C

Step 7. Vortex briefly

Step 8. Add another 40μl of 1M DTT.

Step 9. Add another 15 μl of 10mg/ml proteinase K.

Step 10. Mix gently by inversion.

Step 11. Incubate at 60°C for 2 h or until hair has dissolved completely.

2 hr @ 60 °C

Step 12. Add an equal volµme of chloroform:isoamyl alcohol.

Step 13. Centrifuge at 10,000 g for 10 minutes.

10 min @ 10000 𝘨

Step 14. Transfer the upper aqueous layer into a new microcentrifuge tube.

Step 15. Add 10 μl of 10 mg/ml RNaseA.

Step 16. Incubate at 37 °C for 30 minutes.

30 min @ 37 °C

Step 17. Add an equal volµme of chloroform:isoamyl alcohol.

Step 18. Mix gently by inverting the tube for a few minutes.

Step 19. Centrifuge at 10,000g for 10 minutes.

10 min @ 10000 𝘨

Step 20. Transferred upper aqueous layer into a new microcentrifuge tube.

Step 21. Add twice the volµme of cold isopropanol and 1/10 volµme of 3M sodiµm acetate.

Step 22. Chill at 20°C for 1 hour.

1 hr @ 20 °C

Step 23. Centrifuge at 10,000 g for 10 minutes.

10 min @ 10000 𝘨

Step 24. Discard supernatant.

Step 25. Add 250 μl 70% ethanol

Step 26. Centrifuge at 10,000 g for 10 minutes.

10 min @ 10000 𝘨

Step 27. Discard supernatant.

Step 28. Invert uncapped to air-dry the pellet.

Step 29. Resuspend in 50 μl water.