DTT Protocol

1. Collect hair sample into a 1.5-ml microcentrifuge tube. 2. Add 500μl **digestion buffer**. 3. Add 40μl of **1M DTT**. 4. Add 15μl of 10mg/ml **proteinase K**. 5. Vortex. 6. Incubate overnight at 56°C. 7. Vortex. 8. Add another 40μl of **1M DTT**. 9. Add another 15μl of 10mg/ml **proteinase K**. 10. Mix gently by inversion. 11. Incubate at 60°C for 2 h or until hair has dissolved completely. 12. Add an equal volume of **chloroform:isoamyl alcohol**. 13. Centrifuge at 10,000*g* for 10 min. 14. Transfer the upper aqueous layer into a new microcentrifuge tube. 15. Add 10μl of 10mg/ml **RNaseA**. 16. Incubate at 37°C for 30 min. 17. Add an equal volume of **chloroform:isoamyl alcohol**. 18. Mix gently by inverting the tube for a few minutes. 19. Centrifuge at 10,000*g* for 10 min. 20. Transferred upper aqueous layer into a new microcentrifuge tube. 21. Add twice the volume of cold **isopropanol** and $\frac{1}{10}$ volume of 3M **sodium acetate**. 22. Chill at 20°C for 1 h. 23. Centrifuge at 10,000*g* for 10 min. 24. Discard supernatant. 25. Add 250μl **70% ethanol** 26. Centrifuge at 10,000*g* for 10 min. 27. Discard supernatant. 28. Invert uncapped to air-dry the pellet. 29. Resuspend in 50μl water.

Chelex Protocol

1. Collect hair sample into a 1.5-ml microcentrifuge tube. 2. Add 200μl 5% Chelex solution. 3. Add 10μl of 10mg/mL proteinase K. 4. Incubate at 56◦C overnight. 5. Vortex. 6. Incubate at 100° C for 8 min. 7. Centrifuge at 16,000 × *g* for3 min. 8. Transfer to a new 1.5-mL microcentrifuge tube.

Reagents

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10ml 1M DTT DTT molecular weight: 154.253 g/mol 1. 1.5g of DTT 2. 8 mL of H2O. 3. Adjust the total volume to 10mL Store wrapped in aluminum foil at -20°C. #

Digestion Buffer, pH

7.5 10mM Tris-HCl, 10mM EDTA, 50 mM NaCl, 0.5% SDS To make 50ml total volume: 1. 500μl 5M NaCL 2. 500μl 1M Tris-HCL, pH7.5 3. 1ml 0.5M EDTA 4. 2.5ml 10% SDS 5. Add ddH₂O to 50ml. #

Chloroform

:isoamyl alcohol (24:1) To make 50ml: 1. 48ml chloroform 2. 2ml iso-amyl alcohol