Extraction Buffer (100 ml)
0.14 M NaCl --------------------------------------- 2.8 ml of
5 M NaCl
10 mM Tris-HCl (pH 7.5) -------------------------- 1 ml of 1 M Tris-HCl
1.5 mM MgCl2 ------------------------------------- 0.15 ml of 1 M MgCl2
DEPC-treated H2O -------------------------------- 96.05 ml
Lysis Solution
Guanidinium thiocyanate ---------------------------- 100 g
Distilled H2O --------------------------------------- 117 ml
Sodium citrate (pH 7.0) ----------------------------- 5.25 ml
of 1 M Sodium citrate
10% Sarkosyl -------------------------------------- 10.5 ml
Dissolve at 65oC, and store at room temperature for several months.
Before use, add 72 ul of 2-mercaptoethanol to 10 ml of lysis solution.
11. Add 1.5 ml chloroform: isoamyl alcohol (49:1).
12. Spin at 4,000g for 10 minutes.
13. Take the upper phase, add 5.7 ml of isopropanol, and leave
on ice for 20 minutes.
14. Spin at 4,500 rpm for 30 minutes.
15. Resuspend the pellet in 0.3 ml of Lysis Solution, transfer
to a microfuge tube, and incubate on ice for 15 minutes.
16. Precipitate the RNA with 1 ml of 96% ethanol at -70oC.
17. Spin at full speed for 10 minutes, wash the pellet with 1
ml 70% ethanol, and spin down again.
18. Remove the supernatant and resuspend the pellet in 200 ul
of DEPC-treated H2O.
19. Add the followings for DNase (RNase-free) reaction. 1 M Tris-HCl (pH 7.5) ---------------------- 10 ul 1 M MgCl2 ---------------------------------- 2 ul RNase-free DNase -------------------------- 2 ul RNase Inhibitor ------------------------------ 0.5 ul