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Overview |
With this protocol, transcripts that were initiated from specific genes by RNA polymerases prior to permeabilization can be measured. Instead of a nuclear extract, permeabilized cells are used. |
A. Permeabilization of Cells |
C. Isolation of RNA 1. Resuspend the cell pellet in 0.5 ml of Guanidinium Thiocyanate Buffer. 2. Add 50 μl of 2 M Sodium Acetate, pH 4. Mix thoroughly by inversion. 3. Add 0.5 ml of Phenol. Mix thoroughly by inversion. 4. Add 100 μl of Chloroform:IAA. Mix thoroughly by inversion. 5. Vortex vigorously for 10 sec. 6. Incubate on ice for 15 min. 7. Centrifuge at 10,000 X g for 20 min at 4℃. 8. Transfer the upper aqueous phase containing the RNA to a new microcentrifuge tube. Avoid the white cloudy interface that contains DNA and protein. 9. Add 1 volume of 100% Isopropanol (or 2 volumes of 100% Ethanol). 10. Incubate at -20℃ for at least 1 hr to precipitate the RNA. 11. Centrifuge at 10,000 X g for 20 min at 4℃ to pellet the RNA. 12. Transfer the pellet with a pipet tip or a sterile Pasteur pipet to a new 1.5 ml microfuge tube. 13. Resuspend the pellet in 0.3 ml of Guanidinium Thiocyanate Buffer. Heating to 65 to 70℃ and vortexing will help resuspend the pellet. 14. Add 1 volume of Isopropanol (or 2 volumes of absolute Ethanol). 15. Incubate for 1 hr at -20℃ to precipitate the RNA. 16. Centrifuge at maximum speed (13,000 rpm) in a microcentrifuge for 10 min at 4℃ to pellet the RNA. Remove the supernatant. 17. Add 1 ml of 75% Ethanol to wash the RNA pellet. 18. Centrifuge at maximum speed (13,000 rpm) in a microcentrifuge for 10 min at 4℃ to pellet the RNA. 19. Allow the pellet to air dry. Avoid over-drying the pellet. 20. Resuspend the RNA pellet in DEPC-treated TE or DEPC-treated ddH2 O (see Hint #3). D. Preparation of Slot Blot Membrane for Hybridization (see Hint #4). 1. Add 1 volume of 0.5 N NaOH to the target DNA (see Hint #5). Two μg of DNA are needed for each slot of the slot blot 2. Incubate for 15 min at 85℃ to denature the DNA . 3. Incubate on ice for 5 min. 4. Cut a piece of nitrocellulose to the size of the slot-blot vacuum manifold. 5. Equilibrate the nitrocellulose membrane in 10X SSC. 6. Assemble the slot blot apparatus according to the manufacturer's instructions. 7. To the denatured DNA , add 0.15 volume of 7.5 M Ammonium Acetate and sufficient 10X SSC to dilute the DNA concentration to 10 ng/μl. 8. Add 200 μl of the DNA solution to each of the slots of the slot blot apparatus. 9. Apply a vacuum to the dot blot apparatus to draw the DNA solution through the membrane. 10. Rinse each slot with 10X SSC. 11. Bake the nitrocellulose filter at 80℃ for 2 hr in a vacuum oven to link the DNA to the membrane. |
E. Hybridization of Nitrocellulose Membrane 1. Prehybridize the membrane for 1 hr at 55℃ in Hybridization Solution or a hybridization solution of your choice. 2. Add isolated transcribed RNA (from Step #C20) to the Hybridization Solution the membrane is in. 3. Incubate overnight at 55℃ to hybridize the transcribed RNA to the DNA on the membrane (see Hint #6). 4. Wash the membrane twice for 15 min each in Wash Buffer 1 at room temperature (see Hint #7) 5. Wash the membrane twice for 15 min each in Wash Buffer 2 at 55℃. 6. Expose the film to membrane for autoradiography. 7. Develop film in 3 to 7 days. F. TCA Precipitation to Determine Incorporation of [32 P] GTP into Nucleic Acid (see Hint #8) 1. Add 1 μl aliquot of reaction assay to a 13 x 100 mm tube containing 2 ml of Yeast tRNA Solution. 2. Add 1 ml of 10% TCA. Mix. 3. Incubate on ice for 10 min. 4. Wash Whatman glass microfiber GF/C filters with 5 ml of 5% TCA using a vacuum manifold (with the vacuum on). 5. Pour the sample onto the filter. 6. Rinse the tube with 5% TCA and pour the rinse onto the filter. 7. Repeat Step #F6. 8. Wash the filters twice with approximately 5 ml of 5% TCA. 9. Wash the filters twice with 5 ml of 95% Ethanol 10. Turn off the vacuum and remove the filters. 11. Dry the filters thoroughly at room temperature or in a warm (42℃ to 100℃) oven. 12. Place filters in scintillation vials. 13. Add Scintillation fluid. 14. Count in a scintillation counter. | ||
10 mM UTP | Store 100 μl aliquots at -70℃ Prepare in 10 mM Tris-Cl, pH 7.5 Filter sterilize |
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10 mM CTP | Store 100 μl aliquots at -70℃ Prepare in 10 mM Tris-Cl, pH 7.5 Filter sterilize |
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10 mM ATP | Store 100 μl aliquots at -70℃ Prepare in 10 mM Tris-Cl, pH 7.5 Filter sterilize |
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Phosphocreatine | Prepare fresh Prepare in ddH2 O 100 mg/ml Phosphocreatine, disodium salt; hydrate |
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Yeast tRNA Solution | 50 mM EDTA, pH 8.0 1% (w/v) Yeast tRNA |
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Creatine Phosphokinase (CPK) | Prepare in Transcription Buffer 50% (v/v) Glycerol 0.2% (w/v) Creatine Phosphokinase, Type I Store at -20℃ |
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5% TCA | 5% (v/v) TCA Stock |
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6 mM Magnesium Acetate 20 mM Tris-Cl, pH 8.0 100 mM KCl Filter Sterilize Store at 4℃ 10 mM Ammonium Chloride 0.3 mM EDTA Add 2 μl of 0.5 M DTT per ml of buffer right before use. 10% (v/v) Glycerol |
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10% TCA | 10% (v/v) TCA Stock |
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0.5 M DTT | Store 500 μl aliquots at -20℃ |
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100% TCA Stock | Dissolve in 227 ml ddH2 O 500 g Trichloroacetic Acid (TCA) |
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0.4% Trypan Blue | Prepare in PBS 0.4% (w/v) Trypan Blue |
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Wash Buffer 2 | 0.1X SSC 0.1% SDS |
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1% Lysolecithin | Store at -20℃ 10 mg/ml L-alpha-Lysolecithin, Type I |
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Wash Buffer 1 | 0.1% SDS 2X SSC |
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Permeabilization Buffer | 33 mM Ammonium Chloride 150 mM Sucrose Autoclave 30 mM HEPES, pH 7 Store at 4℃ 4.5 mM Magnesium Acetate 7 mM KCl |
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Denhardt's Solution (100X) | 10 g Ficoll 400 10 g Polyvinylpyrrolidone 10 g Bovine Serum Albumin (Fraction V) Bring final volume to 500 ml with ddH2 O Store at -20℃ in aliquots |
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Hybridization Solution | 1% (w/v) SDS 5X SSC Add DNA just before use. 100 μg/ml Heat-denatured sheared salmon sperm DNA (or Herring testes DNA ) 5X Denhardt's Solution 50% (v/v) Formamide (CAUTION! see Hint #1) |
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SSC (10X) | pH 7.2 0.15 M Sodium Citrate 1.5 M NaCl |
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95% (v/v) Ethanol | ||
PBS | pH 7.2 2.7 mM KCl 4.3 mM Sodium Phosphate Dibasic (Na2 HPO4 ) 1.8 mM Potassium Phosphate Monobasic (KH2 PO4 ) 137 mM NaCl |
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75% (v/v) Ethanol | ||
7.5 M Ammonium Acetate | ||
0.5 M NaOH | ||
Chloroform:IAA | 49:1 Chloroform:Isoamyl Alcohol (CAUTION! see Hint #1) |
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Phenol | Store at 4℃ in a dark glass bottle Water-saturated Phenol (CAUTION! see Hint #1) |
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2 M Sodium Acetate, pH 4 | ||
Guanidinium Thiocyanate Buffer | 4 M Guanidinium Thiocyanate Add the 2-Mercaptoethanol just before use. 0.5% Sarkosyl 25 mM Sodium Citrate, pH 7.0 Can be stored for up to 3 months without the 2-Mercaptoethanol 0.1 M 2-Mercaptoethanol |
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α-Amanitin (1,000X) | Store at -20℃ 2 mg/ml α-Amanitin Prepare in ddH2 O |
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Combined NTPs | 500 μl 10 mM UTP 500 μl 10 mM ATP Store aliquots at -70℃ 500 & |
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