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REAGENTS
Guanadinium Thiocyanate
1.0 M Sodium Citrate pH 7.0
10% Sarcosyl
2-mercaptoethanol
water saturated phenol pH 4.0-7.0
Na2EDTA MOPS （free acid）
2.0 M Sodium Acetate pH 4.0
Formaldehyde
Formamide
10% SDS
49:1 Chloroform:Isoamyl Alcohol
SOLUTIONS
filter sterilize through 0.2 μm filter
NOTE: Solution D may be made ahead of time and stored at room temp for one month,but the 2-mercaptoethanol must be added immediately before use.
PROTOCOL
1） Aspirate off media
2） Lyse cells in Solution D
T-150 = 4 ml Solution D
Transfer to pre-chilled 30 ml Oakridge centrifuge tube; make sure there is enough space for additions below.
3） Sequentially add:
a） 0.1 volume 2.0 M NaOAc - mix well
b） 1 volume phenol - mix well
c） 0.2 volume chlorofom/isoamyl alcohol
4） Vortex 10 sec
5） Incubate on ice for 15 min
6） Centrifuge 10,000 x g, 20 min, 4 ℃
7） Transfer aqueous phase to a new 50 ml conical
8） Precipitate with 1 volume isopropanol, >1 hour, -20 ℃
9） Centrifuge in 50 ml conical tube, 3,000 x g, 1 hour, 4 ℃
alternatively, spin in oakridge tubes 10,000 x g, 20 min, 4 ℃
10） Dissolve pellet in 0.3 ml Solution D; transfer to microfuge tube
11） Precipitate with 1 volume isopropanol, >1 hour, -20 ℃
12） Centrifuge 15 min, 4 ℃
13） Wash pellet with 75% ethanol
14） Dry*, dissolve in H2O**, heat to 65 ℃for 10 min to completely dissolve.
*Do not over dry pellet, otherwise it will be impossible to dissolve
**use approximately 150 μl H2O per T-150 flask
Chomczynske, P. and Sacchi, N. Analytical Biochem. 1987. 162:156-159
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