Differential-Display Reverse Transcription-PCR (DDRT-PCR)

Differential-Display Reverse Transcription-PCR (DDRT-PCR)

Detalles Bibliográficos
Autores principales: Colonna-Romano, Sergio. (Autor), Leone, Antonella. (Autor), Maresca, Bruno. (Autor)
Autor Corporativo: SpringerLink (Online service)
Formato: eBook
Lenguaje:English
Publicado: Berlin, Heidelberg : Springer Berlin Heidelberg : Imprint: Springer, 1998.
Edición:1st ed. 1998.
Colección:Springer Lab Manuals
Materias:
Cell biology.
Biochemistry.
Cell Biology.
Biochemistry, general.
Acceso en línea:https://doi.org/10.1007/978-3-642-80454-0
Tabla de Contenidos:
  • Overview
  • 1 Differential Display of mRNAs: Principles and General Applications
  • 2 DDRT-PCR: A Brief Description
  • 3 Summary
  • 1 Preparation of Total RNA
  • 1.1 Extraction from Yeast by Phenol
  • 1.2 Extraction by Guanidine Thiocyanate, Plant Tissue
  • 1.3 Extraction by Guanidine Thiocyanate, Animal Cells
  • 1.4 Extraction from Fish Liver
  • 1.5 RNA Quantification
  • 1.6 DNase Treatment
  • 1.7 Verification of Integrity
  • 2 Differential Display
  • 2.1 Reverse Transcription of Total RNA
  • 2.2 PCR Amplification
  • 3 Size Separation of cDNA Fragments
  • 3.1 Gel Electrophoresis
  • 3.2 End Labeling of DNA Markers
  • 4 Isolation of Differentially Expressed cDNA Fragments
  • 4.1 Alignment of a Differential-Display Gel Using a Film Image
  • 4.2 Elution of cDNA Fragments from Gel
  • 5 Reamplification of Eluted cDNA
  • 6 Cloning of Amplified cDNA Fragments
  • 6.1 Purification of cDNA Fragments from Agarose
  • 6.2 Ligation of cDNA into Vectors
  • 6.3 Preparation of CaCl2 Competent Cells for Transformation
  • 6.4 Preparation of Electrocompetent Cells
  • 6.5 Transformation of CaCl2 Competent Cells
  • 6.6 Transformation of Competent Cells by Electroporation
  • 7 Checking Subcloned Fragments
  • 8 Confirmation of Differential Expression of Cloned cDNA Fragments
  • 8.1 Slot and Northern Blots
  • 8.2 DNA Labeling and Hybridization
  • 8.3 Ribonuclease Protection Assay
  • 8.4 Nuclear Run-On Assay
  • 9 Northern Blot Affinity Capturing of cDNA
  • 9.1 PCR Labeling of Differentially Expressed cDNA
  • 9.2 Northern Blot Affinity Capturing
  • 10 Sequencing of Differentially Expressed cDNA Fragments
  • 10.1 Plasmid DNA Preparation
  • 10.2 Sequencing Reaction with Sequenase Version 2.0 T7 DNA Polymerase
  • 10.3 Sequencing Reaction with Thermo-Sequenase Cycle-Sequencing Kit
  • Materials, Equipment, Reagents, and Suppliers
  • References.

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