14. TriReagent™ (Sigma-Aldrich) or TRIzol™ Reagent
(Ambion): Store at 4
C.
15. Chloroform: Store at room temperature.
16. Isopropanol: Store at room temperature.
17. 100% Ethanol: Store at room temperature.
18. “Cold” ethanol: 50 mL 100% Ethanol cooled to À20
C .
19. 95% Ethanol: Mix 47.5 mL of 100% ethanol with 2.5 mL of
water. Mix well and store at À20
C.
20. Diethyl pyrocarbonate (DEPC)-treated water: DEPC is toxic;
prepare the solution under a chemical hood. In a bottle, add
0.001% volume of DEPC to 100 mL water. DEPC has limited
solubility in water as evidenced by the appearance of globules;
thus mix well until globules disappear. Incubate for 16 h, and
autoclave. Make aliquots of 2–10 mL, and store at À20
C.
21. 1 M Tris–HCl, pH 7.5: Weigh 12.11 g of Tris and transfer to a
glass beaker. Add about 70 mL of water, mix, and adjust pH to
7.5 with 1 N HCl (see Note 1). Transfer to a glass cylinder and
adjust volume to 100 mL with water. Autoclave and store at
room temperature.
22. 5 M LiCl solution: 5 M LiCl, 50 mM Tris–HCl, pH 7.5. Weigh
21.2 g of LiCl and transfer to a glass beaker. Add 5 mL of 1 M
Tris–HCl, pH 7.5, and water to a volume of 90 mL. Mix
thoroughly until LiCl is completely dissolved. Transfer to a
glass cylinder and adjust volume to 100 mL with water. Autoclave and store at room temperature.
23. 1 M Sodium acetate, pH 4.5: Weigh 8.2 g of sodium acetate
and transfer to a glass beaker. Add water to a volume of 70 mL
and mix until sodium acetate is completely dissolved. Adjust
pH to 4.5 with 99% pure acetic acid. Transfer to a glass cylinder
and adjust volume with water to 100 mL. Autoclave and store
at room temperature.
24. 0.5 M EDTA, pH 8.0: Weigh 29.2 g of ethylenediaminetetraacetic acid (EDTA) and transfer to a glass beaker. Add 150 mL
of water. Mix well and add NaOH pellets until EDTA is
completely dissolved (see Note 2). Adjust pH to 8.0 with 1 N
NaOH. Transfer to a glass cylinder and adjust volume to
200 mL with water. Autoclave and store at room temperature.
25. 2Â Loading buffer: 95% (v/v) Formamide, 20 mM EDTA,
pH 8.0, 0.05% (w/v), bromophenol blue, 0.05% (w/v) xylene
cyanol. Weigh 0.025 g of bromophenol blue and 0.025 g of
xylene cyanol and transfer to a 50 mL tube. Add 2 mL of 0.5 M
EDTA, pH 8.0, and 0.5 mL of water. Mix by vortexing and add
47.5 mL of formamide. Mix well and store at room
temperature.
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Ste ´ phanie Lalande et al.
(Ambion): Store at 4
C.
15. Chloroform: Store at room temperature.
16. Isopropanol: Store at room temperature.
17. 100% Ethanol: Store at room temperature.
18. “Cold” ethanol: 50 mL 100% Ethanol cooled to À20
C .
19. 95% Ethanol: Mix 47.5 mL of 100% ethanol with 2.5 mL of
water. Mix well and store at À20
C.
20. Diethyl pyrocarbonate (DEPC)-treated water: DEPC is toxic;
prepare the solution under a chemical hood. In a bottle, add
0.001% volume of DEPC to 100 mL water. DEPC has limited
solubility in water as evidenced by the appearance of globules;
thus mix well until globules disappear. Incubate for 16 h, and
autoclave. Make aliquots of 2–10 mL, and store at À20
C.
21. 1 M Tris–HCl, pH 7.5: Weigh 12.11 g of Tris and transfer to a
glass beaker. Add about 70 mL of water, mix, and adjust pH to
7.5 with 1 N HCl (see Note 1). Transfer to a glass cylinder and
adjust volume to 100 mL with water. Autoclave and store at
room temperature.
22. 5 M LiCl solution: 5 M LiCl, 50 mM Tris–HCl, pH 7.5. Weigh
21.2 g of LiCl and transfer to a glass beaker. Add 5 mL of 1 M
Tris–HCl, pH 7.5, and water to a volume of 90 mL. Mix
thoroughly until LiCl is completely dissolved. Transfer to a
glass cylinder and adjust volume to 100 mL with water. Autoclave and store at room temperature.
23. 1 M Sodium acetate, pH 4.5: Weigh 8.2 g of sodium acetate
and transfer to a glass beaker. Add water to a volume of 70 mL
and mix until sodium acetate is completely dissolved. Adjust
pH to 4.5 with 99% pure acetic acid. Transfer to a glass cylinder
and adjust volume with water to 100 mL. Autoclave and store
at room temperature.
24. 0.5 M EDTA, pH 8.0: Weigh 29.2 g of ethylenediaminetetraacetic acid (EDTA) and transfer to a glass beaker. Add 150 mL
of water. Mix well and add NaOH pellets until EDTA is
completely dissolved (see Note 2). Adjust pH to 8.0 with 1 N
NaOH. Transfer to a glass cylinder and adjust volume to
200 mL with water. Autoclave and store at room temperature.
25. 2Â Loading buffer: 95% (v/v) Formamide, 20 mM EDTA,
pH 8.0, 0.05% (w/v), bromophenol blue, 0.05% (w/v) xylene
cyanol. Weigh 0.025 g of bromophenol blue and 0.025 g of
xylene cyanol and transfer to a 50 mL tube. Add 2 mL of 0.5 M
EDTA, pH 8.0, and 0.5 mL of water. Mix by vortexing and add
47.5 mL of formamide. Mix well and store at room
temperature.
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