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In&Vitro&Transcription&Assay&(Run-off&Assay)&using&Permeabilized&Cells
来源:生物谷
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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.
Procedure&
A. Permeabilization of Cells 1. Harvest tissue culture cells by trypsinization or scraping into sterile, ice-cold PBS. 2. Count the cell suspension by a cell counter. If clumping is evident (or even suspected), count with a hemacytometer. An accurate cell count is important. 3. Collect cells by centrifugation at 1,000 X g for 5 min at 4°C. 4. Aspire supernatant. Resuspend cell pellet in 10 ml of PBS. 5. Centrifuge at 1,000 X g for 5 min at 4°C. 6. Suspend cell pellet in Permeabilization Buffer to a cell concentration of 108 cells/ml. 7. Check permeabilization by taking 10 μl of the cell suspension and mixing it with Trypan Blue. Determine the percentage of cell viability by recording the percent of cells that exclude the blue dye. 8. Keep the cell suspension on ice. 9. Add one-one hundredth volume of Lysolecithin. Mix gently but thoroughly. 10. Incubate for 1 min at 4°C. 11. Pellet cells at 800 X g in a table-top centrifuge or equivalent for 3 min at 4°C. 12. Remove the supernatant. Resuspend the cells in 1 ml of Permeabilization Buffer. 13. Pellet the cells at 1,500 rpm in a table-top centrifuge for 3 min at 4°C. 14. Resuspend permeabilized cells in Transcription Buffer to a concentration of 4 X 108 cells/ml. 15. Store cells as 250 μl aliquots at -70°C. B. In vitro Transcription Reaction (Run-off) 1. Thaw 1 tube (250 μl, 1 X 108 cells) of permeabilized cells on ice. Suspend gently. 2. If less than 108 cells are to be transcribed, than take desired volume of cells and dilute to 250 μl with Transcription Buffer. 3. Add to the cell suspension 1.5 μl CPK to give a final concentration of 10 μg/ml 7.6 μl of Creatine Phosphate to give a final concentration of 10 mM 20 μl of Combined NTPs to give a final concentration of 220 μM 25 μl of [32P] GTP (&3,000 Ci/mmol) (CAUTION! see Hint #1) The total volume should be 304.1 μl (see Hint #2). 4. Mix gently and incubate at 30°C for 30 min. Vortex 4 or 5 times during the incubation. 5. Take 1 μl aliquots at various time points during the incubation, for example, at 0, 10, 20, and 30 min. 6. Use the 1 μl aliquots to determine the amount of incorporation of label into TCA precipitable counts (see Section F) 7. At the end of the 30 min incubation, centrifuge the samples at 1,500 rpm in a microcentrifuge for 3 min. Discard the supernatant in radioactive waste receptacle. 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°C. 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°C for at least 1 hr to precipitate the RNA. 11. Centrifuge at 10,000 X g for 20 min at 4°C 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°C 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°C to precipitate the RNA. 16. Centrifuge at maximum speed (13,000 rpm) in a microcentrifuge for 10 min at 4°C 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°C 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 ddH2O (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°C 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°C 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°C 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°C 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°C. 6. Expose the film to membrane for autoradiography. 7. Develop film in 3 to 7 days. F. TCA Precipitation to Determine Incorporation of [32P] 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°C to 100°C) oven. 12. Place filters in scintillation vials. 13. Add Scintillation fluid. 14. Count in a scintillation counter.
Store 100 μl aliquots at -70°C Prepare in 10 mM Tris-Cl, pH 7.5 Filter sterilize
Store 100 μl aliquots at -70°C Prepare in 10 mM Tris-Cl, pH 7.5 Filter sterilize
Store 100 μl aliquots at -70°C Prepare in 10 mM Tris-Cl, pH 7.5 Filter sterilize
Phosphocreatine
Prepare fresh Prepare in ddH2O 100 mg/ml Phosphocreatine, hydrate
Yeast tRNA Solution
50 mM EDTA, pH 8.0 1% (w/v) Yeast tRNA
Creatine Phosphokinase (CPK)
Prepare in Transcription Buffer 50% (v/v) Glycerol 0.2% (w/v) Creatine Phosphokinase, Type I Store at -20°C
5% (v/v) TCA Stock
Transcription Buffer
6 mM Magnesium Acetate 20 mM Tris-Cl, pH 8.0 100 mM KCl Filter Sterilize Store at 4°C 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
10% (v/v) TCA Stock
Store 500 μl aliquots at -20°C
100% TCA Stock
Dissolve in 227 ml ddH2O 500 g Trichloroacetic Acid (TCA)
0.4% Trypan Blue
Prepare in PBS 0.4% (w/v) Trypan Blue
Wash Buffer 2
0.1X SSC 0.1% SDS
1% Lysolecithin
Store at -20°C 10 mg/ml L-alpha-Lysolecithin, Type I
Wash Buffer 1
0.1% SDS 2X SSC
Permeabilization Buffer
33 mM Ammonium Chloride 150 mM Sucrose Autoclave 30 mM HEPES, pH 7 Store at 4°C 4.5 mM Magnesium Acetate 7 mM KCl
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 ddH2O Store at -20°C in aliquots
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)
pH 7.2 0.15 M Sodium Citrate 1.5 M NaCl
95% (v/v) Ethanol
pH 7.2 2.7 mM KCl 4.3 mM Sodium Phosphate Dibasic (Na2HPO4) 1.8 mM Potassium Phosphate Monobasic (KH2PO4) 137 mM NaCl
75% (v/v) Ethanol
7.5 M Ammonium Acetate
0.5 M NaOH
Chloroform:IAA
49:1 Chloroform:Isoamyl Alcohol (CAUTION! see Hint #1)
Store at 4°C in a dark glass bottle Water-saturated Phenol (CAUTION! see Hint #1)
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
α-Amanitin (1,000X)
Store at -20°C 2 mg/ml α-Amanitin Prepare in ddH2O
Combined NTPs
500 μl 10 mM UTP 500 μl 10 mM ATP Store aliquots at -70°C 500 μl 10 mM CTP
Greater than 3,000 Ci/mmol (CAUTION! see Hint #1)
95% (v/v) Ethanol
BioReagents and Chemicals
Sodium Hydroxide CTP UTP Polyvinyl Pyrolidone Tris Magnesium Acetate Formamide Sodium Phosphate, Dibasic Ammonium Acetate Potassium Chloride Glass Microfiber GF/C filters Sodium Chloride Nitrocellulose Membrane DTT α-Amanitin Scintillation Fluid [32P]-GTP Sarkosyl Sucrose DNA, Salmon Sperm Glycerol Trichloroacetic Acid (TCA) Guanidinium Thiocyanate Phosphocreatine, Disodium S Hydrate RNase A Creatine Phosphokinase, Type I L-α-Lysolecithin, Type I Ethanol Isoamyl Alcohol 2-Mercaptoethanol Chloroform Ammonium Chloride Phenol Isopropanol ATP SDS HEPES Bovine Serum Albumin (BSA), Fraction V Ficoll 400 Sodium Acetate Trypan Blue EDTA Potassium Phosphate, Monobasic Sodium Citrate
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions. 2. To measure the level of RNA polymerase II activity, i.e. hnRNA synthesis, add 1 μl of alpha-amanitin to a final concentration of 2 μg/ml. 3. To minimize degradation of RNA by RNAses, wear gloves when handling samples and reagents and change gloves regularly while working. For more information and tips, see "Working with RNA" in the Reference Pages. 4. For each reaction, prepare DNA slots with the DNA(s) of interest and a negative control of plasmid DNA. 5. The in vitro transcribed RNA (from Section B) is detected by hybridization to target DNA. 6. Hybridization up to 72 hr is fine. 7. To decrease background signal, add RNAse A to 1 mg/ml to the Wash Buffer 1. 8. Counts incorporated into nucleic acid are precipitated by TCA. [32P]-GTP is not precipitated.
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人乳头瘤病毒L1基因在酵母体外翻译系统和原代角质细胞中的表达研究
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