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, 44 ℃
alternatively, spin in oakridge tubes 10,000 x g, 20 min, 4 4 ℃
10) Dissolve pellet in 0.3 ml Solution D; transfer to microfuge tube
11) Precipitate with 1 volume isopropanol, >1 hour, -20 4 ℃
12) Centrifuge 15 min, 4 4 ℃
13) Wash pellet with 75% ethanol
14) Dry*, dissolve in H2O**, heat to 65 4 ℃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
