REAGENTSGuanadinium 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.

PROTOCOL1） 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