Editing Trizol (section)

== Protocol 1  ==

1. Harvest cells, spin down cells gently, add an approximately equal pellet volume of 1X PBS and suspend cells<br>2. If you freeze cell pellet on dry ice, do not add 1XPBS, add Trizol directly to frozen pellet, then mix vigerously.<br>3. Add 1ml TRI Reagent (for between 10^5 and 3 x 10^7 cells) and suspend cells immediately.<br>4. Incubate at room temperature for 5-15 minutes.<br>5. Add 200ul CHCl3, vortex vigorously for 10-20 seconds.<br>6. Incubate at room temperature for 5 minutes.<br>7. Spin in at maximum speed for 15 minutes.<br>8. Transfer aqueous phase into a new tube<br>9. Add 0.5ml isopropanol, mix, and keep at room temperature for 10 minutes.<br>10. Spin at max speed for 20 minutes.<br>11. Take off (or aspirate) the aqueous/isopropanol solution.<br>12. Wash with 800 ul 70% EtOH (stored at â20oC)[i.e. add 70% EtOH, vortex, spin 5 minutes (high speed in microfuge) and take off EtOH]<br>13. Dry in speedy vac and suspend RNA in 50-250 ul molecular biology grade H2O. 

'''Homemade Trizol Reagent '''(Acid Guanidinium Thiocyanate-Phenol-Chloroform) solution 

- 4M Guanidinium thiocyanate: add 293 ml H2O to 250 g guanidinium thiocyanate (Fluka)<br>- 0.75 M Sodium citrate pH 7.0<br>- 2 M Sodium Acetate pH 4.0<br>- 10% Sarcosyl (1g/10ml H2O and filter)<br>- Phenol (nucleic acid grade, Sigma) (warm phenol bottle at 65 oC, add 0.1% w/v hydroxyquinoline and saturate with H2O) 

'''21 ml of TRIzol reagent '''<br>- Mix 10 ml 4M guanidinium thiocyanate, 352ul 0.75M sodium citrate (pH 7.0) and 528ul 10% sarcosyl<br>- Add 76 µl 14.3 M Ã-mercaptoethanol (Sigma), total final volume is 10.9 ml.<br>- Place 10 ml of this mixture in a new tube, add 1 ml 2M sodium acetate (pH 4.0) and 10 ml water saturated phenol. 

'''105 ml of TRIzol reagent'''<br>- Mix 50 ml 4M guanidinium thiocyanate, 1.76 ml 0.75M sodium citrate (pH 7.0) and 2.64ml 10% sarcosyl<br>- Add 381 µl 14.3 M Ã-mercaptoethanol (Sigma), total final volume is 54.5 ml.<br>- Place 50 ml of this mixture in a new tube, add 5 ml 2M sodium acetate (pH 4.0) and 50 ml water saturated phenol. 

Store TRIzol reagent at 4 oC. It is stable for at least 2 months. 

'''1L Trizol''' 

Reagents Volume/mass Final concentration<br>Acid Phenol 380mL 38%<br>Guanidine thiocyanate 94.53g 0.8M<br>Ammonium thiocyanate 30.45g 0.4M<br>Sodium acetate, pH 5.0 33.4mL of 3M stock 0.1M<br>Glycerol 50mL 5%<br>RNase-free water Adjust final volume to 1L 

'''Other reagents:'''<br>DEPC-Water Adjust the final volume to 1 L<br>0.8 M sodium citrate / 1.2 M NaCl<br>Isopropanol (2-Propanol)<br>Chloroform<br>DEPC-Water<br>75% ethanol prepared with DEPC-Water<br> 

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