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Human RNA Extraction protocol(哈佛RNA提取方法)

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2069

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) Drundefined, dissolve in H2undefined~E_heat_to_65__℃for 10 min to completely dissolve.

Chomczynske, P. and Sacchi, N. Analytical Biochem. 1987. 162:156-159

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