Editing Trizol (section)

== Protocol 2  ==

Material 

TRIzol Reagent. 

0.8 M sodium citrate / 1.2 M NaCl 

Isopropanol (2-Propanol) 

Chloroform 

DEPC-Water 

75% ethanol prepared with DEPC-Water 

RNase Inhibitor (e.g., aseERASE, BIO 101 Cat. 2601-104)<br>50 mL sterile plastic screw-cap centrifuge tubes 

1. Grind 1g tissue in liquid nitrogen in a mortar and pestle. 

2. Transfer powdered tissue to a 50 mL sterile plastic screw-cap centrifuge tube containing 15 ml TRIzol reagent. Incubate samples at room temperature or at 60°C for 5 min. 

3. Homogenize tissue with homogenizer for 15 seconds. Repeat once. 

4. Centrifuge samples at 12,000 x g at 4°C for 10 min. 

5. Transfer supernatant into new sterile 50 mL sterile plastic screw-cap centrifuge tube. Discard pellet. 

6. Add 3 mL chloroform to each tube in hood. Shake tubes vigorously with vortex for 15 sec. 

7. Let tubes sit at room temp 2-3 min. Centrifuge tubes at 10,000 x g at 4°C for 15 min. 

8. Carefully pipet aqueous phase into a clean screw-cap centrifuge tube; discard interphase and lower phase into waste. 

9. Precipitate RNA by adding isopropanol and 0.8 M sodium citrate/1.2 M NaCl, half volume of the aqueous phase each. Cover tube and mix by gentle inversion. Let sit at room temperature for 10 min. 

10. Centrifuge tubes at 10,000 x g at 4°C for 10 min. Discard supernatant. 

11. Wash pellet with 20 ml of 75% ethanol. Vortex briefly. 

12. Centrifuge at 10,000 x g at 4°C for 10 min. Discard supernatant; briefly dry pellet on kimwipe. 

13. Add 100-250 µL DEPC-Water, to pellet. Resuspend RNA by pipetting up and down a few times. 

14. Add 1 µL RNase inhibitor aseERASE to a 250 µL RNA sample. If having problems resuspending the RNA pellet, we suggest incubation at 55 - 60°C for 10 min. 

15. Transfer sample to microcentrifuge tube at room temperature. 

16. Spin samples at high speed in microcentrifuge tube for 5 min at room temperature (to pellet the material that would not resuspend). 

17. Transfer RNA solution (supernatant) to a new tube. Determine RNA concentration and quality by spectrophotometry. 

Note: For optimal spectrophotometric measurements, RNA aliquots should be diluted with water or buffer with a basic pH. Water with pH&lt; 7.5 falsely decreases the 260/280 ratio.<br> 

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'''Reagents''' 

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'''Volume/mass''' 

| 
'''Final concentration''' 

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Acid Phenol 

| 
380mL 

| 
38% 

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Guanidine thiocyanate 

| 
94.53g, or 118.16 g? 

| 
0.8M 

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Ammonium thiocyanate 

| 
30.45g, or 76.12 g? 

| 
0.4M 

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| 
Sodium acetate, pH 5.0 

| 
33.4mL of 3M stock 

| 
0.1M 

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| 
Glycerol 

| 
50mL 

| 
5% 

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| 
DEPC treated RNase-free water 

| 
Adjust final volume to 1L 

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