| Description |
1 online resource (lviii, 413 pages) : colour illustrations |
| Series |
Methods in enzymology, 0076-6879 ; v. 530 |
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Methods in enzymology ; v. 530
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| Contents |
Contents note continued: 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 Immobilize Ribosomes on mRNA in Cells -- 6.Step 2 Prepare Cell Lysate for Polysome Analysis -- 7.Step 3 Size Fractionation of Polysomes -- References -- 11.Polysome Analysis for Determining mRNA and Ribosome Association in Saccharomyces cerevisiae / Jeff Coller -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 Grow Yeast Cells for Polysome Analysis -- 6.Step 2 Prepare Sucrose Gradient -- 7.Step 3 Cell Lysis and Sedimentation Analysis -- 8.Step 4 Fractionate the Sucrose Gradient -- References -- Source References -- Section V Reverse Transcription -- 12.0ligo(dT)-primed RT-PCR Isolation of Polyadenylated RNA Degradation Intermediates / Gadi Schuster -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 RNA Purification -- 6.Step 2 Oligo(dT)-Primed Reverse Transcription -- 7.Step 3 First Round PCR (PCR #1) -- 8.Step 4 Gel Purification of PCR Products -- |
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Contents note continued: 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 Separate RNA by Denaturing Agarose Gel Electrophoresis -- 6.Step 2 Transfer RNA from the Gel to a Nylon Membrane -- 7.Step 3 Radiolabel the Probe and Hybridize it to the Membrane -- References -- 4.Explanatory Chapter: Nuclease Protection Assays / Elizabeth Eyler -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 Hybridization of Labeled Probes and RNA Sample -- 6.Step 2 Nuclease Digestion -- 7.Step 3 Nuclease Inactivation and Probe Recovery -- 8.Step 4 Electrophoretic Separation and Detection -- 9.Interpretation and Troubleshooting -- References -- Section II In Vitro Transcription -- 5.In Vitro Transcription from Plasmid or PCR-amplified DNA / Rachel Green -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 Phenol Extract DNA Template -- 6.Step 2 T7 RNA Polymerase in vitro Transcription Reaction -- 7.Step 3 Phenol Extraction of Transcription Reaction -- |
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Contents note continued: 15.Step 11a Plasmid DNA Isolation and DNA Sequencing -- 16.Step 8b Radiolabel a Primer and Use it in PCR -- 17.Step 9b Analyze PCR Products by Polyacrylamide Gel Electrophoresis -- References -- Source References -- Section VI RNA Labeling -- 14.RNA Radiolabeling / Daniel Herschlag -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 5'-Dephosphorylation of Transcribed RNAs Using Shrimp Alkaline Phosphatase and 5'-Labeling with T4 Polynucleotide Kinase -- 6.Step 2 Dephosphorylation of RNAs with 2', 3'-Cyclic Phosphates Using T4 Polynucleotide Kinase Followed by Phenol/Chloroform Extraction -- 7.Step 3'-Labeling with E coli DNA Polymerase Klenow Fragment -- 8.Step 4 Preparing the Denaturing Polyacrylamide Gel -- 9.Step 5 PAGE Purification and RNA Extraction -- References -- 15.Fluorescently Labeling Synthetic RNAs / Daniel Herschlag -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 Precipitate Unlabeled Oligonucleotide -- |
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Contents note continued: 16.Step 2(IV) Viewing Results of Partition Function Calculation -- 17.Step 3(IV) Annotating Secondary Structures with Base Pair Probabilities -- 18.Protocol V Folding and Aligning RNAs: Dynalign -- 19.Step 1(V) Load Sequences for Dynalign Calculation -- 20.Step 2(V) (Optional) Adding Constraints to Dynalign -- 21.Step 3(V) Submit Sequences for Dynalign Calculation -- 22.Step 4(V) View Results of a Dynalign Calculation -- 23.Protocol VI Aligning and Folding RNAs: RNAalifold -- 24.Step 1(VI) Assemble RNA Sequence Data -- 25.Step 2(VI) Construct RNA Sequence Alignment -- 26.Step 3(VI) Submit Aligned Sequences to the RNAalifold (or Consensus Secondary Structure Prediction -- 27.Step 4(VI) Visualizing the Results of an RNAalifold Calculation -- 28.Protocol VII Finding Common RNA Shapes: RNAshapes -- 29.Step 1(VII) Prepare Sequences for Submission to RNAshapes (See Protocol II, Steps 1.1-1.6, Save the Sequences in Fasta Format) -- |
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Contents note continued: 30.Step 2(VII) Set Up and Run RNAshapes Calculation -- 31.Protocol VIII Searching for Similar RNA Structures: The RNA Strand Database -- 32.Step 1(VIII) Navigate to RNA Strand Page and Set Up Search -- 33.Step 2(VIII) Analyze the Results of RNA Strand Search -- 34.Step 3(VIII) Search Rfam Database -- 35.Step 4(V1II) Analyze Rfam Results -- 36.Protocol IX Predicting RNA Pseudoknots: pknotsRG -- 37.Step 1 (IX) Prepare Sequences for Submission to pknotsRG (See Protocol II, Steps 1.1-1.6, Save the Sequences in Fasta Format) -- 38.Step 2(IX) Set Up and Run pknotsRG Calculation -- 39.Step 3(IX) Visualizing the Results of pknotsRG Calculation -- Acknowledgments -- References -- 2.Reverse-Transcription PCR (RT-PCR) / Julia Bachman -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 Unwinding RNA -- 6.Step 2 Reverse Transcription Reaction -- 7.Step 3 PCR Reaction -- 8.Step 4 Analysis of Product -- References -- 3.Northern Blotting / Rachel Green -- |
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Contents note continued: 6.Step 2 Determine the Absorbance Value -- 7.Step 3 Use Beer's Law to Calculate the Sample Concentration -- References -- 19.RNA Purification - Precipitation Methods / Jon Lorsch -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocols -- 5.Method 1 Ethanol Precipitation of RNA -- 6.Method 2 Lithium Chloride Precipitation -- References -- Section VIII RNA Sequencing -- 20.Reverse Transcriptase Dideoxy Sequencing of RNA / Jon Lorsch -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 Label Primer -- 6.Step 2 Anneal Primer -- 7.Step 3 Primer Extension and Chain Termination -- 8.Step 4 Resolution of Labeled Products by Urea-PAGE -- References -- Section IX RNA Structure Experimental Analysis -- 21.RNA Structure Experimental Analysis - Chemical Modification / Gloria Culver -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 Modification of the Complex -- 6.Step 2 RNA Extraction -- |
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Contents note continued: 6.Step 2 Test-Labeling Reactions (Optional) -- 7.Step 3 Large-Scale Labeling Reaction and Gel Purification -- References -- Section VII RNA Purification -- 16.Analysis of RNA by Analytical Polyacrylamide Gel Electrophoresis / Joseph D. Puglisi -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 Preparing the Gel -- 6.Step 2 Running the Gel -- 7.Step 3 Visualizing the RNA -- References -- 17.RNA Purification by Preparative Polyacrylamide Gel Electrophoresis / Joseph D. Puglisi -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 Preparing the Gel -- 6.Step 2 Running the Gel -- 7.Step 3 Visualizing the RNA -- 8.Step 4A RNA Extraction Using the ̀Crush and Soak' Method -- 9.Step 4B RNA Extraction by Electroelution -- References -- 18.Explanatory Chapter: Nucleic Acid Concentration Determination / Jon Lorsch -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 Determine Parameters to Use in Beer's Law -- |
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Contents note continued: 7.Step 3 Quantification and Normalization of RNA -- 8.Step 4 Primer Extension -- References -- 22.Structural Analysis of RNA Backbone Using In-Line Probing / Rachel Green -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 in vitro RNA Transcription -- 6.Step 2 Labeling RNA at 5' Terminus with 32P -- 7.Step 3 In-line Probing Reaction -- References |
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Contents note continued: 8.Step 4 Column Purification of RNA Transcript -- References -- Source References -- Section III Measurement of RNA Synthesis and Decay Rates -- 6.Measurement of In Vivo RNA Synthesis Rates / Jo Ann Wise -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 Preparation of Immobilized DNA or RNA -- 6.Step 2 In-Vivo 32P-Labeling of Nascent Transcripts -- 7.Step 3 Isolation of Total RNA -- 8.Step 4 Hybridization of Labeled RNA to Immobilized Probes -- References -- Source References -- 7.Method for Measuring mRNA Decay Rate in Saccharomyces cerevisiae / Jeff Coller -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 Turn Off mRNA Transcription and Remove Samples of Yeast at Various Time Points -- 6.Step 2 RNA Extraction from Yeast Cells -- 7.Step 3 Formaldehyde Agarose Denaturing Gel Electrophoresis and Northern Transfer -- 8.Step 4 Prepare a Radiolabeled Oligonucleotide Probe -- |
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Contents note continued: 9.Step 5 Hybridize Probe to Membrane to Detect the Reporter mRNA -- 10.Step 6 Strip the Membrane -- 11.Step 7 Hybridize the Membrane to a Probe to Detect SCR1 RNA -- 12.Step 8 Calculate the mRNA Half-Life -- References -- Source References -- Section IV Polysome Analysis -- 8.Analysis of Polysomes from Bacteria / Kurt Fredrick -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Purified Water -- 5.Protocol -- 6.Step 1 Preparation of Sucrose Gradients -- 7.Step 2 Preparation of Cell Lysates -- 8.Step 3 Sedimentation of Components of the Lysate Through Sucrose Gradients -- 9.Step 4 Pump Gradients -- References -- 9.Polysome Profile Analysis - Yeast / Leos Valasek -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 Grow Yeast Cultures -- 6.Step 2 Preparation of Yeast Whole Cell Extracts -- 7.Step 3 Polysome Profile Analysis using Sucrose Density Gradient Centrifugation -- References -- 10.Polysome Analysis of Mammalian Cells / Rachel Green -- |
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Contents note continued: 9.Step 5 Second Round PCR (PCR #2) -- 10.Step 6 Gel Purification -- 11.Step 7 T/A Clone the PCR Products -- 12.Step 8 Electroporation and Plating -- 13.Step 9 Colony Screening by PCR -- 14.Step 10 Plasmid DNA Isolation and DNA Sequencing -- References -- Source References -- 13.Circularized RT-PCR (cRT-PCR): Analysis of the 5' Ends, 3' Ends, and poly(A) Tails of RNA / Gadi Schuster -- 1.Theory -- 2.Equipment -- 3.Materials -- 4.Protocol -- 5.Step 1 RNA Purification -- 6.Step 2 Circularization of RNA Molecules -- 7.Step 3 Reverse Transcription Using a Gene-Specific Reverse Primer (R1) -- 8.Step 4 PCR Using Forward Primer (F1) and Reverse Primer (R2) -- 9.Step 5 Gel Purification of PCR Products -- 10.Step 6 Second Round PCR -- 11.Step 7 Gel Purification of PCR Products -- 12.Step 8a T/A Clone the Second Round PCR Products -- 13.Step 9a Electroporate and Plate Bacteria -- 14.Step 10a Screen Clones by Colony PCR -- |
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Machine generated contents note: Section I RNA Protocols -- 1.Computational Prediction of RNA Secondary Structure / Walter N. Moss -- 1.Theory -- 2.Equipment -- 3.Protocol I Searching for Homologous RNA Sequences: BLAST -- 4.Step 1 (I) Submit Sequence for BLAST Analysis -- 5.Step 2(l) Analyze BLAST Results -- 6.Protocol II Single Sequence RNA Folding -- 7.Step 1 (II) Prepare Sequence for Submission to RNAstructure -- 8.Step 2(II) Submit Sequence to RNAstructure -- 9.Step 3(II) viewing the Results of Secondary Structure Calculation -- 10.Protocol III Single Sequence RNA Folding Including Constraints -- 11.Step 1 (III) Enter Constraints for Single Sequence Structure Prediction -- 12.Step 2(III) Submit Sequence to RNAstructure -- 13.Step 3(III) Viewing the Results of Secondary Structure Calculation -- 14.Protocol IV Assessing the Quality of a Prediction: The Partition Function Calculation -- 15.Step 1 (IV) Calculate the Secondary Structure Partition Function -- |
| Bibliography |
Includes bibliographical references and index |
| Notes |
Online version of the print title |
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Mode of access: World Wide Web |
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System requirements: Internet connectivity, World Wide Web browser and Adobe Acrobat reader |
| Subject |
Enzymology.
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RNA.
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| Form |
Electronic book
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| Author |
Lorsch, Jon.
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| ISBN |
0124200370 |
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9780124200371 |
