Lecture 15 - Blotting Techniques | Plant Biotechnology - Botany PDF Download

 Page 1


                    Blotting Techniques 
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
Lesson Prepared Under MHRD project “ National Mission 
on Education Through ICT” 
 
Discipline: Botany 
Paper: Plant Biotechnology 
 
National Coordinator: Prof. S.C. Bhatla 
 
Lesson: Blotting Techniques 
Department/College: Department of Genetics, University of Delhi, 
South Campus 
 
Lesson Developer: Vinee Khanna 
 
Lesson Reviewer: Dr Manoj  K. Sharma 
Department/College : School of Biotechnology 
Jawaharlal Nehru University 
 
Language Editor: Namrata Dhaka 
Department/College: Department of Genetics, University of Delhi, 
South Campus 
 
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL 
 
 
 
Page 2


                    Blotting Techniques 
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
Lesson Prepared Under MHRD project “ National Mission 
on Education Through ICT” 
 
Discipline: Botany 
Paper: Plant Biotechnology 
 
National Coordinator: Prof. S.C. Bhatla 
 
Lesson: Blotting Techniques 
Department/College: Department of Genetics, University of Delhi, 
South Campus 
 
Lesson Developer: Vinee Khanna 
 
Lesson Reviewer: Dr Manoj  K. Sharma 
Department/College : School of Biotechnology 
Jawaharlal Nehru University 
 
Language Editor: Namrata Dhaka 
Department/College: Department of Genetics, University of Delhi, 
South Campus 
 
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL 
 
 
 
                    Blotting Techniques 
 
Institute of Lifelong Learning, University of Delhi 
Table of Contents 
Chapter: Blotting methods 
? Introduction 
? Southern blotting 
? Introduction 
? Principle 
? Methodology 
? Preparation of genomic DNA 
? DNA separation 
? Blotting methods 
? DNA-DNA hybridization 
? Detection 
? Applications 
? Limitations 
? Northern Blotting 
? Introduction 
? Procedure 
? Preparation of RNA 
? DNA separation 
? Blotting methods 
? DNA-DNA hybridization 
? Detection 
? Applications 
? Advantages 
? Disadvantages 
? Western Blotting 
? Introduction 
? Principle 
? Procedure 
? Sample preparation 
? Separation on gel 
? Transfer to the blotting membrane 
? Detection 
Page 3


                    Blotting Techniques 
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
Lesson Prepared Under MHRD project “ National Mission 
on Education Through ICT” 
 
Discipline: Botany 
Paper: Plant Biotechnology 
 
National Coordinator: Prof. S.C. Bhatla 
 
Lesson: Blotting Techniques 
Department/College: Department of Genetics, University of Delhi, 
South Campus 
 
Lesson Developer: Vinee Khanna 
 
Lesson Reviewer: Dr Manoj  K. Sharma 
Department/College : School of Biotechnology 
Jawaharlal Nehru University 
 
Language Editor: Namrata Dhaka 
Department/College: Department of Genetics, University of Delhi, 
South Campus 
 
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL 
 
 
 
                    Blotting Techniques 
 
Institute of Lifelong Learning, University of Delhi 
Table of Contents 
Chapter: Blotting methods 
? Introduction 
? Southern blotting 
? Introduction 
? Principle 
? Methodology 
? Preparation of genomic DNA 
? DNA separation 
? Blotting methods 
? DNA-DNA hybridization 
? Detection 
? Applications 
? Limitations 
? Northern Blotting 
? Introduction 
? Procedure 
? Preparation of RNA 
? DNA separation 
? Blotting methods 
? DNA-DNA hybridization 
? Detection 
? Applications 
? Advantages 
? Disadvantages 
? Western Blotting 
? Introduction 
? Principle 
? Procedure 
? Sample preparation 
? Separation on gel 
? Transfer to the blotting membrane 
? Detection 
                    Blotting Techniques 
 
Institute of Lifelong Learning, University of Delhi 
? Applications 
? Glossary 
? Exercise 
? References 
? Further reading   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Page 4


                    Blotting Techniques 
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
Lesson Prepared Under MHRD project “ National Mission 
on Education Through ICT” 
 
Discipline: Botany 
Paper: Plant Biotechnology 
 
National Coordinator: Prof. S.C. Bhatla 
 
Lesson: Blotting Techniques 
Department/College: Department of Genetics, University of Delhi, 
South Campus 
 
Lesson Developer: Vinee Khanna 
 
Lesson Reviewer: Dr Manoj  K. Sharma 
Department/College : School of Biotechnology 
Jawaharlal Nehru University 
 
Language Editor: Namrata Dhaka 
Department/College: Department of Genetics, University of Delhi, 
South Campus 
 
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL 
 
 
 
                    Blotting Techniques 
 
Institute of Lifelong Learning, University of Delhi 
Table of Contents 
Chapter: Blotting methods 
? Introduction 
? Southern blotting 
? Introduction 
? Principle 
? Methodology 
? Preparation of genomic DNA 
? DNA separation 
? Blotting methods 
? DNA-DNA hybridization 
? Detection 
? Applications 
? Limitations 
? Northern Blotting 
? Introduction 
? Procedure 
? Preparation of RNA 
? DNA separation 
? Blotting methods 
? DNA-DNA hybridization 
? Detection 
? Applications 
? Advantages 
? Disadvantages 
? Western Blotting 
? Introduction 
? Principle 
? Procedure 
? Sample preparation 
? Separation on gel 
? Transfer to the blotting membrane 
? Detection 
                    Blotting Techniques 
 
Institute of Lifelong Learning, University of Delhi 
? Applications 
? Glossary 
? Exercise 
? References 
? Further reading   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                    Blotting Techniques 
 
Institute of Lifelong Learning, University of Delhi 
 
Learning Outcomes 
 
The following chapter would help you to familiarize with the following: 
? Methodologies involved in DNA, RNA and protein sample preparations for 
hybridization. 
? Blotting methods and transfer membranes used. 
? Different methods of detection. 
? Applications of the various blotting methods. 
? Advantages and disadvantages of the various blotting techniques. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Page 5


                    Blotting Techniques 
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
Lesson Prepared Under MHRD project “ National Mission 
on Education Through ICT” 
 
Discipline: Botany 
Paper: Plant Biotechnology 
 
National Coordinator: Prof. S.C. Bhatla 
 
Lesson: Blotting Techniques 
Department/College: Department of Genetics, University of Delhi, 
South Campus 
 
Lesson Developer: Vinee Khanna 
 
Lesson Reviewer: Dr Manoj  K. Sharma 
Department/College : School of Biotechnology 
Jawaharlal Nehru University 
 
Language Editor: Namrata Dhaka 
Department/College: Department of Genetics, University of Delhi, 
South Campus 
 
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL 
 
 
 
                    Blotting Techniques 
 
Institute of Lifelong Learning, University of Delhi 
Table of Contents 
Chapter: Blotting methods 
? Introduction 
? Southern blotting 
? Introduction 
? Principle 
? Methodology 
? Preparation of genomic DNA 
? DNA separation 
? Blotting methods 
? DNA-DNA hybridization 
? Detection 
? Applications 
? Limitations 
? Northern Blotting 
? Introduction 
? Procedure 
? Preparation of RNA 
? DNA separation 
? Blotting methods 
? DNA-DNA hybridization 
? Detection 
? Applications 
? Advantages 
? Disadvantages 
? Western Blotting 
? Introduction 
? Principle 
? Procedure 
? Sample preparation 
? Separation on gel 
? Transfer to the blotting membrane 
? Detection 
                    Blotting Techniques 
 
Institute of Lifelong Learning, University of Delhi 
? Applications 
? Glossary 
? Exercise 
? References 
? Further reading   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                    Blotting Techniques 
 
Institute of Lifelong Learning, University of Delhi 
 
Learning Outcomes 
 
The following chapter would help you to familiarize with the following: 
? Methodologies involved in DNA, RNA and protein sample preparations for 
hybridization. 
? Blotting methods and transfer membranes used. 
? Different methods of detection. 
? Applications of the various blotting methods. 
? Advantages and disadvantages of the various blotting techniques. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                    Blotting Techniques 
 
Institute of Lifelong Learning, University of Delhi 
 
 
Introduction 
 
The oldest method of blotting was given by Edwin Southern in the year 1973 (refer to the 
figure). In this method the DNA was fragmented with endonucleases and run on an agarose 
gel. The separated bands were then transferred to a nylon membrane using the capillary 
action method. The radiolabelled probes were hybridized to the membrane and analyzed on 
the photographic film. This method is the basis of all the blotting methods available today. 
Each of the methods has been modified according to the sample analyzed and to increase 
the efficiency of the data obtained. 
Blotting involves four major steps: 
? Separation of sample by gel electrophoresis. 
? Immobilization on a solid support. 
? Binding of a probe on the target. 
? Detection and analysis of the target by visualization.