Editing Trizol

== 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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== 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> 

{| cellspacing="0" cellpadding="0" border="1" align="left"
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'''Reagents''' 

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'''Volume/mass''' 

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'''Final concentration''' 

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Acid Phenol 

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380mL 

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38% 

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Guanidine thiocyanate 

| 
94.53g, or 118.16 g? 

| 
0.8M 

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Ammonium thiocyanate 

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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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|}

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== Protocol 3  ==

'''Solution D''' (based on Chomczynski and Sacchi[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=2440339 1987]/[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=17406285 2006]) is: 

*4 M guanidinium thiocyanate 
*25 mM sodium citrate 
*pH 7.0 
*0.5% (wt/vol) N-laurosylsarcosine (Sarkosyl) 
*0.1 M 2-mercaptoethanol

Prepare stock with: 

*dissolving 250 g guanidinium thiocyanate in 293 ml water at 65 Ã°C 
*add 17.6 ml of 0.75 M sodium citrate, pH 7.0 
*26.4 ml of 10% (wt/vol) N-laurosylsarcosine

(stored &lt;3 months at room temperature) 

Working solution from stock: 

*add 0.36 ml of 98% 2-mercaptoethanol to 50 ml of stock solution

(store &lt;1 month at RT)


'''Using Solution D'''

Assuming you start with 10 million cells or 100 mg of tissue (keep cool when possible): 

*add 1 ml solution D (don't linger on this step) 
*transfer to tubes 
*add 0.1 ml of 2 M sodium acetate, pH 4.0, and invert tube to mix 
*add 1 ml water-saturated phenol (never buffered phenol) and invert tube 
*add 0.2 ml of chloroform/isoamyl alcohol (49:1) and shake vigorously for 10 sec 
*centrifuge 20min 10000G 4ÃºC 
*transfer top aqueous phase into new tube 
*precipitate

[[Category:Protocols]]