Southern, northern, and western blot protocols are similar, and begin with electrophoretic separation of protein and nucleic acid fragments on a gel, which are then transferred to a membrane (nitrocellulose membrane, polyvinylidene difluoride (PVDF) membrane, etc.) where they are immobilized.
Southern blotting is a hybridization technique for identification of particular size of DNA from the mixture of other similar molecules. This technique is based on the principle of separation of DNA fragments by gel electrophoresis and identified by labelled probe hybridization.
Southern blotting is a technique used to detect specific DNA sequences. It involves digesting DNA samples with restriction enzymes, separating the fragments via gel electrophoresis, transferring the fragments to a membrane, and using a labeled probe to detect sequences that hybridize to the membrane. The size of DNA fragments can be determined by comparing them to markers of known sizes. Southern blotting has applications in gene mapping, evolution studies, and disease diagnosis.
This document discusses different blotting techniques used to detect DNA, RNA, and proteins. Southern blotting is used to detect DNA and was developed by Edwin Southern in 1975. It involves separating DNA fragments by size, transferring them to a membrane, and using a probe to detect specific sequences. Northern blotting detects RNA and was developed in 1979. It similarly separates RNA and uses a probe to detect sequences of interest. Western blotting detects proteins and was developed in 1981. It separates proteins by SDS-PAGE gel electrophoresis, transfers them to a membrane, and uses an antibody probe to detect the target protein. These techniques are widely used in research and diagnostics.
Southern Blotting and Related DNA Detection Techniques MD ASIQUR RAHMAN
The document describes Southern blotting, a technique developed by Edwin Southern in 1975. It involves transferring DNA fragments separated by electrophoresis onto a membrane, where they can be detected through hybridization with labeled probes. Specifically, DNA is extracted, digested with restriction enzymes, separated on a gel, and transferred to a membrane via capillary action. The membrane-bound DNA can then be probed to detect specific fragments through hybridization and visualized. The Southern blot technique allows detection of a targeted DNA fragment against a complex background and has various applications in research, forensics, and medicine.
1. DNA or proteins are separated by gel electrophoresis.
2. The molecules are then transferred to a membrane through either southern or western blotting.
3. For southern blotting, DNA is detected through hybridization with a labeled probe. For western blotting, specific proteins are detected through interaction with labeled antibodies.
4. These techniques are used for diagnostic purposes and research applications such as identifying DNA sequences or proteins of interest.
1. DNA or proteins are separated by gel electrophoresis.
2. The molecules are then transferred to a membrane through blotting.
3. For Southern blotting, DNA is detected using labeled probes that hybridize to complementary DNA sequences. For Western blotting, proteins are detected using primary and secondary antibodies that bind to the protein of interest.
4. These techniques are used for applications like identifying genes, detecting infectious diseases and genetic disorders, and forensic analysis.
Southern, Northern, and Western blotting techniques allow researchers to detect specific DNA, RNA, and protein sequences, respectively. Southern blotting involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, and using a probe to identify specific sequences. Northern blotting is similar but detects RNA, and Western blotting detects proteins using antibodies. These techniques are used for applications like gene mapping, diagnostics, studying gene expression, and confirming transgenic organisms.
Southern blotting is a hybridization technique for identification of particular size of DNA from the mixture of other similar molecules. This technique is based on the principle of separation of DNA fragments by gel electrophoresis and identified by labelled probe hybridization.
Southern blotting is a technique used to detect specific DNA sequences. It involves digesting DNA samples with restriction enzymes, separating the fragments via gel electrophoresis, transferring the fragments to a membrane, and using a labeled probe to detect sequences that hybridize to the membrane. The size of DNA fragments can be determined by comparing them to markers of known sizes. Southern blotting has applications in gene mapping, evolution studies, and disease diagnosis.
This document discusses different blotting techniques used to detect DNA, RNA, and proteins. Southern blotting is used to detect DNA and was developed by Edwin Southern in 1975. It involves separating DNA fragments by size, transferring them to a membrane, and using a probe to detect specific sequences. Northern blotting detects RNA and was developed in 1979. It similarly separates RNA and uses a probe to detect sequences of interest. Western blotting detects proteins and was developed in 1981. It separates proteins by SDS-PAGE gel electrophoresis, transfers them to a membrane, and uses an antibody probe to detect the target protein. These techniques are widely used in research and diagnostics.
Southern Blotting and Related DNA Detection Techniques MD ASIQUR RAHMAN
The document describes Southern blotting, a technique developed by Edwin Southern in 1975. It involves transferring DNA fragments separated by electrophoresis onto a membrane, where they can be detected through hybridization with labeled probes. Specifically, DNA is extracted, digested with restriction enzymes, separated on a gel, and transferred to a membrane via capillary action. The membrane-bound DNA can then be probed to detect specific fragments through hybridization and visualized. The Southern blot technique allows detection of a targeted DNA fragment against a complex background and has various applications in research, forensics, and medicine.
1. DNA or proteins are separated by gel electrophoresis.
2. The molecules are then transferred to a membrane through either southern or western blotting.
3. For southern blotting, DNA is detected through hybridization with a labeled probe. For western blotting, specific proteins are detected through interaction with labeled antibodies.
4. These techniques are used for diagnostic purposes and research applications such as identifying DNA sequences or proteins of interest.
1. DNA or proteins are separated by gel electrophoresis.
2. The molecules are then transferred to a membrane through blotting.
3. For Southern blotting, DNA is detected using labeled probes that hybridize to complementary DNA sequences. For Western blotting, proteins are detected using primary and secondary antibodies that bind to the protein of interest.
4. These techniques are used for applications like identifying genes, detecting infectious diseases and genetic disorders, and forensic analysis.
Southern, Northern, and Western blotting techniques allow researchers to detect specific DNA, RNA, and protein sequences, respectively. Southern blotting involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, and using a probe to identify specific sequences. Northern blotting is similar but detects RNA, and Western blotting detects proteins using antibodies. These techniques are used for applications like gene mapping, diagnostics, studying gene expression, and confirming transgenic organisms.
This document discusses various blotting techniques used to detect and analyze biomolecules like DNA, RNA, and proteins. It describes the Southern blot technique developed by Edwin Southern used to detect specific DNA sequences. It also discusses the Northern blot technique used to detect RNA, developed by James Alwine and George Stark. Finally, it summarizes the Western blot technique used to detect specific proteins by using antibodies, developed in 1981. These blotting techniques allow separation and detection of biomolecules through transfer and hybridization/binding reactions.
1. Blotting techniques such as Southern, Northern, and Western blotting allow for the transfer of DNA, RNA, and proteins from a gel to a membrane for detection.
2. The Southern blot detects DNA using hybridization with a labeled probe. The Northern blot detects RNA and the Western blot detects proteins using antibodies.
3. These techniques separate biomolecules by size then transfer and detect them on a membrane using probes or antibodies, allowing analysis of complex samples.
Blotting
A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier.
The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane.
Types of blotting techniques
Southern Blotting
Northern Blotting
Western Blotting
A Southern blot is a method used
in molecular biology for detection of a specific DNA sequence in DNA samples.
Southern blotting combines transfer of electrophoresis -separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.
The method is named after its inventor, the British biologist Edwin Mellor Southern.
- Methods in Southern blotting
- Advantages and disadvantages
Principle and application of blotting techniquesJayeshRajput7
This document discusses various blotting techniques used in molecular biology including Northern blotting, Southern blotting, dot blotting, colony hybridization, and plaque hybridization. Northern blotting is used to detect RNA, Southern blotting detects specific DNA sequences, dot blotting detects proteins without separation, colony hybridization screens bacterial colonies for desired genes, and plaque hybridization identifies recombinant phages. These techniques allow for detection and analysis of nucleic acids and proteins to study gene expression, mutations, genetic diseases, and more.
Blotting techniques involve the separation (via electrophoresis) and transfer of DNA, RNA, or proteins onto a blotting membrane. This separation is generally followed by complexing of the target with a labeled molecule for detection.
Used extensively in analysis of:
DNA.
RNA.
Proteins.
Principle and applications of blotting techniquesJayeshRajput7
The document discusses various blotting techniques used in molecular biology including Northern blotting, Southern blotting, dot blotting, colony hybridization, and plaque hybridization.
Northern blotting involves separating RNA samples by size, transferring them to a membrane, and using a probe to detect specific sequences. Southern blotting is used to detect specific DNA sequences by separating DNA fragments, transferring them to a membrane, and using probes. Dot blotting simplifies the detection of proteins by applying samples directly to a membrane. Colony hybridization screens bacterial colonies for genes of interest by transferring DNA to a membrane and using probes. Plaque hybridization identifies recombinant phages using a similar process to colony hybridization.
Blotting technique including Southern , Northern and Western blotting Rohit Mondal
he given ppt contains all the blotting techniques which is being studied by students in Biotechnology related subject and this PPT contais all blotting techniques in a very elaborative concise manner includes procedure principle application etc so which itwould help any bio student to take proper knowledge in this topic. I hope you will enjoy the content of the topic and would be able to grasp the topic properly
This document discusses various blotting techniques used to detect specific DNA, RNA, and protein molecules. It describes Southern blotting for detecting DNA, Northern blotting for detecting RNA, and Western blotting for detecting proteins. Southern blotting involves separating DNA fragments by gel electrophoresis, transferring them to a membrane, and using a labeled probe for detection. Northern blotting is similar but used for detecting specific RNA sequences. Western blotting uses SDS-PAGE gel electrophoresis to separate proteins, transfers them to a membrane, and detects them using primary and secondary antibodies. These techniques allow detection of specific biomolecules among many contaminants and have various applications in research and diagnostics.
Concept: reannealing nucleic acids to identify sequence of interest.
Separates DNA/RNA in an agarose gel, then detects specific bands using probe and hybridization.
Hybridization takes advantage of the ability of a single stranded DNA or RNA molecule to find its complement, even in the presence of large amounts of unrelated DNA.
Allows detection of specific bands (DNA fragments or RNA molecules) that have complementary sequence to the probe.
Size bands and quantify abundance of molecule.
Blotting techniques such as Southern blotting, Northern blotting, and Western blotting allow researchers to detect specific DNA, RNA, and protein sequences by transferring them from a gel to a membrane and using probes to identify the targets. Southern blotting is used to detect DNA, Northern blotting detects RNA, and Western blotting identifies proteins. These techniques play important roles in research areas like gene expression analysis, disease diagnosis, forensics, and more by providing sensitive and specific detection of biomolecules.
This document provides an overview of blotting techniques, including transfer methods, membranes used, and applications of Southern blotting, Northern blotting, and Western blotting. It discusses how blotting involves transferring proteins, DNA, or RNA from a gel onto a membrane. It describes the capillary, electro-blotting, and vacuum blot transfer processes and membranes like nitrocellulose, nylon, and PVDF. It then explains the specific methods and applications of Southern blotting for detecting DNA, Northern blotting for detecting RNA, and Western blotting for detecting proteins.
This document discusses three biotechnology techniques: DNA microarray, gene sequencing, and SDS-PAGE. It provides details on the principles, methods, and steps for each technique. DNA microarray allows analysis of gene expression for thousands of genes using DNA spots on a solid surface. Gene sequencing determines the order of genes along a chromosome using methods like directed sequencing and shotgun libraries. SDS-PAGE separates molecules by size using polyacrylamide gel and SDS to neutralize protein charge.
This document discusses blotting techniques, specifically Southern blotting. It defines Southern blotting as a technique developed by Professor Sir Edwin Southern in 1975 to detect specific DNA sequences. The technique involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, then using a labeled probe to detect complementary DNA sequences via hybridization. Southern blotting is used for applications like gene discovery, mapping, diagnostics, and DNA fingerprinting.
Nucleic acid hybridization is a technique used to identify specific DNA sequences. It involves denaturing DNA or RNA samples and probes, followed by annealing of the probes to complementary sequences. There are two main types: Southern blotting separates DNA fragments by gel electrophoresis before hybridization with probes, while Northern blotting separates RNA this way. Both techniques allow detection of specific sequences through the use of labeled probes.
The document discusses various blotting techniques used to transfer and detect DNA, RNA, and proteins, including Southern blotting for DNA, Northern blotting for RNA, and Western blotting for proteins. Southern blotting involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, and using a probe to detect specific sequences. It allows researchers to identify genes, map genomes, and study evolution and disease.
Southern blotting detects specific DNA fragments using restriction enzymes and radioactive probes. Northern blotting separates mRNA to detect gene expression at the mRNA level using DNA probes. Western blotting separates proteins using SDS-PAGE gel electrophoresis then detects specific proteins using antibodies in an antigen-antibody reaction. These techniques allow detection of specific DNA, RNA, or protein targets from samples.
Blotting techniques such as Southern blotting, Northern blotting, and Western blotting allow researchers to identify specific molecules like DNA, RNA, and proteins that have been separated in a gel. The process involves transferring molecules from a gel onto a membrane, where they are immobilized. Probes are then used to detect and visualize the target molecules on the membrane. These techniques have various applications, including DNA analysis, gene expression studies, disease diagnosis, and protein analysis. However, non-specific binding can sometimes generate false positives that techniques aim to reduce.
Southern blotting is a technique used to detect specific DNA sequences. It involves separating DNA fragments by size via gel electrophoresis, transferring them to a membrane, and using a labeled probe to hybridize to the target sequence. The probe binds only to complementary DNA fragments, allowing their detection and location on the membrane via autoradiography. Key steps include restriction enzyme digestion of DNA, gel electrophoresis, membrane transfer, probe hybridization, and x-ray film development to visualize hybridized fragments. Southern blotting is used for applications like mutation detection, gene rearrangement studies, and forensic analysis.
Blotting techniques such as Southern blotting, Northern blotting, and Western blotting allow for the detection of specific DNA, RNA, and protein sequences by transferring them from a separation gel onto a membrane and using probes to detect the targets. Southern blotting detects DNA using DNA probes, Northern blotting detects RNA using RNA or DNA probes, and Western blotting detects proteins using antibodies. These techniques are used for applications like detecting gene expression, RNA splicing, and confirming diseases.
Biotechnology is technology that utilizes biological systems, living organisms or parts of this to develop or create different products. Brewing and baking bread are examples of processes that fall within the concept of biotechnology (use of yeast (= living organism) to produce the desired product).
DNA consists of a linear string of nucleotides, or bases, for simplicity, referred to by the first letters of their chemical names--A, T, C and G. The process of deducing the order of nucleotides in DNA is called DNA sequencing. Since the DNA sequence confers information that the cell uses to make RNA molecules and proteins, establishing the sequence of DNA is key for understanding how genomes work. The technology for DNA sequencing was made faster and less expensive as a part of the Human Genome Project. And recent developments have profoundly increased the efficiency of DNA sequencing even further.
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Similar to Southern, Western & Northern Bloting.pdf
This document discusses various blotting techniques used to detect and analyze biomolecules like DNA, RNA, and proteins. It describes the Southern blot technique developed by Edwin Southern used to detect specific DNA sequences. It also discusses the Northern blot technique used to detect RNA, developed by James Alwine and George Stark. Finally, it summarizes the Western blot technique used to detect specific proteins by using antibodies, developed in 1981. These blotting techniques allow separation and detection of biomolecules through transfer and hybridization/binding reactions.
1. Blotting techniques such as Southern, Northern, and Western blotting allow for the transfer of DNA, RNA, and proteins from a gel to a membrane for detection.
2. The Southern blot detects DNA using hybridization with a labeled probe. The Northern blot detects RNA and the Western blot detects proteins using antibodies.
3. These techniques separate biomolecules by size then transfer and detect them on a membrane using probes or antibodies, allowing analysis of complex samples.
Blotting
A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier.
The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane.
Types of blotting techniques
Southern Blotting
Northern Blotting
Western Blotting
A Southern blot is a method used
in molecular biology for detection of a specific DNA sequence in DNA samples.
Southern blotting combines transfer of electrophoresis -separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.
The method is named after its inventor, the British biologist Edwin Mellor Southern.
- Methods in Southern blotting
- Advantages and disadvantages
Principle and application of blotting techniquesJayeshRajput7
This document discusses various blotting techniques used in molecular biology including Northern blotting, Southern blotting, dot blotting, colony hybridization, and plaque hybridization. Northern blotting is used to detect RNA, Southern blotting detects specific DNA sequences, dot blotting detects proteins without separation, colony hybridization screens bacterial colonies for desired genes, and plaque hybridization identifies recombinant phages. These techniques allow for detection and analysis of nucleic acids and proteins to study gene expression, mutations, genetic diseases, and more.
Blotting techniques involve the separation (via electrophoresis) and transfer of DNA, RNA, or proteins onto a blotting membrane. This separation is generally followed by complexing of the target with a labeled molecule for detection.
Used extensively in analysis of:
DNA.
RNA.
Proteins.
Principle and applications of blotting techniquesJayeshRajput7
The document discusses various blotting techniques used in molecular biology including Northern blotting, Southern blotting, dot blotting, colony hybridization, and plaque hybridization.
Northern blotting involves separating RNA samples by size, transferring them to a membrane, and using a probe to detect specific sequences. Southern blotting is used to detect specific DNA sequences by separating DNA fragments, transferring them to a membrane, and using probes. Dot blotting simplifies the detection of proteins by applying samples directly to a membrane. Colony hybridization screens bacterial colonies for genes of interest by transferring DNA to a membrane and using probes. Plaque hybridization identifies recombinant phages using a similar process to colony hybridization.
Blotting technique including Southern , Northern and Western blotting Rohit Mondal
he given ppt contains all the blotting techniques which is being studied by students in Biotechnology related subject and this PPT contais all blotting techniques in a very elaborative concise manner includes procedure principle application etc so which itwould help any bio student to take proper knowledge in this topic. I hope you will enjoy the content of the topic and would be able to grasp the topic properly
This document discusses various blotting techniques used to detect specific DNA, RNA, and protein molecules. It describes Southern blotting for detecting DNA, Northern blotting for detecting RNA, and Western blotting for detecting proteins. Southern blotting involves separating DNA fragments by gel electrophoresis, transferring them to a membrane, and using a labeled probe for detection. Northern blotting is similar but used for detecting specific RNA sequences. Western blotting uses SDS-PAGE gel electrophoresis to separate proteins, transfers them to a membrane, and detects them using primary and secondary antibodies. These techniques allow detection of specific biomolecules among many contaminants and have various applications in research and diagnostics.
Concept: reannealing nucleic acids to identify sequence of interest.
Separates DNA/RNA in an agarose gel, then detects specific bands using probe and hybridization.
Hybridization takes advantage of the ability of a single stranded DNA or RNA molecule to find its complement, even in the presence of large amounts of unrelated DNA.
Allows detection of specific bands (DNA fragments or RNA molecules) that have complementary sequence to the probe.
Size bands and quantify abundance of molecule.
Blotting techniques such as Southern blotting, Northern blotting, and Western blotting allow researchers to detect specific DNA, RNA, and protein sequences by transferring them from a gel to a membrane and using probes to identify the targets. Southern blotting is used to detect DNA, Northern blotting detects RNA, and Western blotting identifies proteins. These techniques play important roles in research areas like gene expression analysis, disease diagnosis, forensics, and more by providing sensitive and specific detection of biomolecules.
This document provides an overview of blotting techniques, including transfer methods, membranes used, and applications of Southern blotting, Northern blotting, and Western blotting. It discusses how blotting involves transferring proteins, DNA, or RNA from a gel onto a membrane. It describes the capillary, electro-blotting, and vacuum blot transfer processes and membranes like nitrocellulose, nylon, and PVDF. It then explains the specific methods and applications of Southern blotting for detecting DNA, Northern blotting for detecting RNA, and Western blotting for detecting proteins.
This document discusses three biotechnology techniques: DNA microarray, gene sequencing, and SDS-PAGE. It provides details on the principles, methods, and steps for each technique. DNA microarray allows analysis of gene expression for thousands of genes using DNA spots on a solid surface. Gene sequencing determines the order of genes along a chromosome using methods like directed sequencing and shotgun libraries. SDS-PAGE separates molecules by size using polyacrylamide gel and SDS to neutralize protein charge.
This document discusses blotting techniques, specifically Southern blotting. It defines Southern blotting as a technique developed by Professor Sir Edwin Southern in 1975 to detect specific DNA sequences. The technique involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, then using a labeled probe to detect complementary DNA sequences via hybridization. Southern blotting is used for applications like gene discovery, mapping, diagnostics, and DNA fingerprinting.
Nucleic acid hybridization is a technique used to identify specific DNA sequences. It involves denaturing DNA or RNA samples and probes, followed by annealing of the probes to complementary sequences. There are two main types: Southern blotting separates DNA fragments by gel electrophoresis before hybridization with probes, while Northern blotting separates RNA this way. Both techniques allow detection of specific sequences through the use of labeled probes.
The document discusses various blotting techniques used to transfer and detect DNA, RNA, and proteins, including Southern blotting for DNA, Northern blotting for RNA, and Western blotting for proteins. Southern blotting involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, and using a probe to detect specific sequences. It allows researchers to identify genes, map genomes, and study evolution and disease.
Southern blotting detects specific DNA fragments using restriction enzymes and radioactive probes. Northern blotting separates mRNA to detect gene expression at the mRNA level using DNA probes. Western blotting separates proteins using SDS-PAGE gel electrophoresis then detects specific proteins using antibodies in an antigen-antibody reaction. These techniques allow detection of specific DNA, RNA, or protein targets from samples.
Blotting techniques such as Southern blotting, Northern blotting, and Western blotting allow researchers to identify specific molecules like DNA, RNA, and proteins that have been separated in a gel. The process involves transferring molecules from a gel onto a membrane, where they are immobilized. Probes are then used to detect and visualize the target molecules on the membrane. These techniques have various applications, including DNA analysis, gene expression studies, disease diagnosis, and protein analysis. However, non-specific binding can sometimes generate false positives that techniques aim to reduce.
Southern blotting is a technique used to detect specific DNA sequences. It involves separating DNA fragments by size via gel electrophoresis, transferring them to a membrane, and using a labeled probe to hybridize to the target sequence. The probe binds only to complementary DNA fragments, allowing their detection and location on the membrane via autoradiography. Key steps include restriction enzyme digestion of DNA, gel electrophoresis, membrane transfer, probe hybridization, and x-ray film development to visualize hybridized fragments. Southern blotting is used for applications like mutation detection, gene rearrangement studies, and forensic analysis.
Blotting techniques such as Southern blotting, Northern blotting, and Western blotting allow for the detection of specific DNA, RNA, and protein sequences by transferring them from a separation gel onto a membrane and using probes to detect the targets. Southern blotting detects DNA using DNA probes, Northern blotting detects RNA using RNA or DNA probes, and Western blotting detects proteins using antibodies. These techniques are used for applications like detecting gene expression, RNA splicing, and confirming diseases.
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Biotechnology is technology that utilizes biological systems, living organisms or parts of this to develop or create different products. Brewing and baking bread are examples of processes that fall within the concept of biotechnology (use of yeast (= living organism) to produce the desired product).
DNA consists of a linear string of nucleotides, or bases, for simplicity, referred to by the first letters of their chemical names--A, T, C and G. The process of deducing the order of nucleotides in DNA is called DNA sequencing. Since the DNA sequence confers information that the cell uses to make RNA molecules and proteins, establishing the sequence of DNA is key for understanding how genomes work. The technology for DNA sequencing was made faster and less expensive as a part of the Human Genome Project. And recent developments have profoundly increased the efficiency of DNA sequencing even further.
DNA sequencing refers to the general laboratory technique for determining the exact sequence of nucleotides, or bases, in a DNA molecule. The sequence of the bases (often referred to by the first letters of their chemical names: A, T, C, and G) encodes the biological information that cells use to develop and operate. Establishing the sequence of DNA is key to understanding the function of genes and other parts of the genome. There are now several different methods available for DNA sequencing, each with its own characteristics, and the development of additional methods represents an active area of genomics research.
The DNA microarray is a tool used to determine whether the DNA from a particular individual contains a mutation in genes like BRCA1 and BRCA2. The chip consists of a small glass plate encased in plastic. Some companies manufacture microarrays using methods similar to those used to make computer microchips.
The three important techniques of biotechnology are: (1) Recombinant DNA Technology (Genetic Engineering) (2) Plant Tissue Culture and (3) Transgenic (Genetically Modified Organisms).
Western blotting is a technique used to detect specific proteins in a tissue sample. It involves three main steps - separating proteins by size through gel electrophoresis, transferring them to a membrane, and detecting the target protein using primary and secondary antibodies. The target protein band can then be visualized, allowing identification and quantification of proteins in a complex mixture. Western blotting is commonly used to identify proteins, estimate their size and amount, and diagnose conditions by detecting antibodies against specific antigens.
Animal Tissue Culture
The foundation of animal cell and tissue culture was laid by Jolly (1903) when he showed that animal cells could not only survive but could divide in culture medium. The actual beginning of animal cell culture and tissue culture was made by Harrison (1907) and later by Carrel (1912) who used frog’s tissue in tissue culture. They successfully showed that animal cells can be grown indefinitely in culture medium just like microorganisms. Later tissues from warm blooded animals like chick and mammals were used as material for tissue culture purpose.
The document provides a history of the development of animal cell culture from the late 19th century to the late 20th century. It describes key developments like the use of antibiotics to prevent contamination, techniques for subculture of adherent cells using trypsin, and the development of standardized culture media. The requirements for an animal cell culture laboratory are outlined, including sterile work areas, incubation facilities, microscopes, tissue culture supplies, and various equipment. The types of culture media, including natural and synthetic options, are also summarized.
Plasmids were the first vectors used in gene cloning. They are natural, self-replicating DNA molecules found in bacteria, eukaryotes, and archaea with high copy numbers and antibiotic resistance genes. Examples include pBR322 and pUC18. Bacteriophages like lambda and M13 are more efficient for cloning large DNA inserts, as they can package up to 53kb of DNA. Phagemids are artificial vectors that combine features of plasmids and M13 phage, allowing blue-white screening. Bacterial artificial chromosomes are similar to E.coli plasmid vectors but derived from F' plasmids, allowing accommodation of large DNA sequences without risk.
Assisted reproductive technology treats infertility and the treatment involves both a man's sperm and a woman's egg. The procedure begins by extracting eggs from a woman's body and then fusing it with the sperm to generate embryos. This embryo is then transferred back into the woman's body.
This document provides a multiple choice quiz on basic laboratory techniques related to blotting. It contains 33 questions testing understanding of techniques like Southern blotting, which is used to analyze the compositional properties of DNA, and transfers DNA from agarose gels to membranes. The questions cover topics like the purpose of blotting, what membranes are used, how large DNA fragments require longer transfer times, and what probes are commonly used in Southern blotting.
Principle of DNA Microarray Technique
The principle of DNA microarrays lies on the hybridization between the nucleic acid strands.
The property of complementary nucleic acid sequences is to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs.
DNA fingerprinting is a technique used to identify and analyze the variations in various individuals at the level of DNA. DNA fingerprinting involves identifying differences in some specific regions in DNA sequence called as repetitive DNA because, in these sequences, a small stretch of DNA is repeated many times.
Biotechnology is defined as the broad area of biology which uses both the technology and the application of living organisms and their components to develop, modify and produce useful products for human welfare. The term ‘Biotechnology’ was coined in the year 1919 by an agricultural engineer Karoly Ereky, hence he is called as the father of Biotechnology.
Principles of Biotechnology
According to modern Biotechnology, the main principles of Biotechnology are:
Genetic engineering, which is used to modify the DNA of the target organism, thereby changing the phenotype of the organism.
Bioprocess engineering, which is the maintenance of sterile conditions to support the growth of large quantities of desired microbes and other eukaryotic cells which are used for the production of new or modified biotechnological products such as antibiotics, enzymes, vaccines, etc.
The techniques of genetic engineering mainly include:
DNA fragment is isolated from the donor organism.
It is inserted into the vector DNA.
It is transferred into an appropriate host.
Cloning of the recombinant DNA in the host organism.
DNA sequencing is a laboratory technique used to determine the exact sequence of bases (A, C, G, and T) in a DNA molecule. The DNA base sequence carries the information a cell needs to assemble protein and RNA molecules. DNA sequence information is important to scientists investigating the functions of genes.
In medicine, DNA sequencing is used for a range of purposes, including diagnosis and treatment of diseases. In general, sequencing allows health care practitioners to determine if a gene or the region that regulates a gene contains changes, called variants or mutations, that are linked to a disorder.
The DNA microarray is a tool used to determine whether the DNA from a particular individual contains a mutation in genes like BRCA1 and BRCA2. The chip consists of a small glass plate encased in plastic. Some companies manufacture microarrays using methods similar to those used to make computer microchips.
A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot contains picomoles of a specific DNA sequence, known as probes.
This chapter provides an overview of DNA microarrays. Microarrays are a technology in which 1000’s of nucleic acids are bound to a surface and are used to measure the relative concentration of nucleic acid sequences in a mixture via hybridization and subsequent detection of the hybridization events. We first cover the history of microarrays and the antecedent technologies that led to their development. We then discuss the methods of manufacture of microarrays and the most common biological applications. The chapter ends with a brief discussion of the limitations of microarrays and discusses how microarrays are being rapidly replaced by DNA sequencing technologies.
Boyd 2014 [16] Record linkage is the process of bringing together data relating to the same individual from within and between different datasets. When a unique person-based identifier exists, linkage can be achieved by simply merging datasets on the identifier.
Linkage is the close association of genes or other DNA sequences on the same chromosome. The closer two genes are to each other on the chromosome, the greater the probability that they will be inherited together.
The two different types of linkage are:
Complete linkage.
Incomplete linkage.
“Linkage and recombination are the phenomena that describe the inheritance of genes.”
Epistasis is a phenomenon in genetics in which the effect of a gene mutation is dependent on the presence or absence of mutations in one or more other genes, respectively termed modifier genes. In other words, the effect of the mutation is dependent on the genetic background in which it appears.
An example of epistasis is the interaction between hair colour and baldness. A gene for total baldness would be epistatic to one for blond hair or red hair. The hair-colour genes are hypostatic to the baldness gene. The baldness phenotype supersedes genes for hair colour, and so the effects are non-additive.
Various types of epistatic gene interaction are 1) Recessive epitasis (9:3:4) 2) Dominant epistasis (12:3:1) 3) Dominant and recessive (inhibitory) epistasis (13:3) 4) Duplicate recessive epistasis (9:7) 5) Duplicate dominant epistasis (15:1) and 6) Polymeric gene interaction (9:6:1).
A review of the growth of the Israel Genealogy Research Association Database Collection for the last 12 months. Our collection is now passed the 3 million mark and still growing. See which archives have contributed the most. See the different types of records we have, and which years have had records added. You can also see what we have for the future.
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আমাদের সবার জন্য খুব খুব গুরুত্বপূর্ণ একটি বই ..বিসিএস, ব্যাংক, ইউনিভার্সিটি ভর্তি ও যে কোন প্রতিযোগিতা মূলক পরীক্ষার জন্য এর খুব ইম্পরট্যান্ট একটি বিষয় ...তাছাড়া বাংলাদেশের সাম্প্রতিক যে কোন ডাটা বা তথ্য এই বইতে পাবেন ...
তাই একজন নাগরিক হিসাবে এই তথ্য গুলো আপনার জানা প্রয়োজন ...।
বিসিএস ও ব্যাংক এর লিখিত পরীক্ষা ...+এছাড়া মাধ্যমিক ও উচ্চমাধ্যমিকের স্টুডেন্টদের জন্য অনেক কাজে আসবে ...
Leveraging Generative AI to Drive Nonprofit InnovationTechSoup
In this webinar, participants learned how to utilize Generative AI to streamline operations and elevate member engagement. Amazon Web Service experts provided a customer specific use cases and dived into low/no-code tools that are quick and easy to deploy through Amazon Web Service (AWS.)
This document provides an overview of wound healing, its functions, stages, mechanisms, factors affecting it, and complications.
A wound is a break in the integrity of the skin or tissues, which may be associated with disruption of the structure and function.
Healing is the body’s response to injury in an attempt to restore normal structure and functions.
Healing can occur in two ways: Regeneration and Repair
There are 4 phases of wound healing: hemostasis, inflammation, proliferation, and remodeling. This document also describes the mechanism of wound healing. Factors that affect healing include infection, uncontrolled diabetes, poor nutrition, age, anemia, the presence of foreign bodies, etc.
Complications of wound healing like infection, hyperpigmentation of scar, contractures, and keloid formation.
How to Build a Module in Odoo 17 Using the Scaffold MethodCeline George
Odoo provides an option for creating a module by using a single line command. By using this command the user can make a whole structure of a module. It is very easy for a beginner to make a module. There is no need to make each file manually. This slide will show how to create a module using the scaffold method.
How to Add Chatter in the odoo 17 ERP ModuleCeline George
In Odoo, the chatter is like a chat tool that helps you work together on records. You can leave notes and track things, making it easier to talk with your team and partners. Inside chatter, all communication history, activity, and changes will be displayed.
Walmart Business+ and Spark Good for Nonprofits.pdfTechSoup
"Learn about all the ways Walmart supports nonprofit organizations.
You will hear from Liz Willett, the Head of Nonprofits, and hear about what Walmart is doing to help nonprofits, including Walmart Business and Spark Good. Walmart Business+ is a new offer for nonprofits that offers discounts and also streamlines nonprofits order and expense tracking, saving time and money.
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Spark Good (walmart.com/sparkgood) is a charitable platform that enables nonprofits to receive donations directly from customers and associates.
Answers about how you can do more with Walmart!"
LAND USE LAND COVER AND NDVI OF MIRZAPUR DISTRICT, UPRAHUL
This Dissertation explores the particular circumstances of Mirzapur, a region located in the
core of India. Mirzapur, with its varied terrains and abundant biodiversity, offers an optimal
environment for investigating the changes in vegetation cover dynamics. Our study utilizes
advanced technologies such as GIS (Geographic Information Systems) and Remote sensing to
analyze the transformations that have taken place over the course of a decade.
The complex relationship between human activities and the environment has been the focus
of extensive research and worry. As the global community grapples with swift urbanization,
population expansion, and economic progress, the effects on natural ecosystems are becoming
more evident. A crucial element of this impact is the alteration of vegetation cover, which plays a
significant role in maintaining the ecological equilibrium of our planet.Land serves as the foundation for all human activities and provides the necessary materials for
these activities. As the most crucial natural resource, its utilization by humans results in different
'Land uses,' which are determined by both human activities and the physical characteristics of the
land.
The utilization of land is impacted by human needs and environmental factors. In countries
like India, rapid population growth and the emphasis on extensive resource exploitation can lead
to significant land degradation, adversely affecting the region's land cover.
Therefore, human intervention has significantly influenced land use patterns over many
centuries, evolving its structure over time and space. In the present era, these changes have
accelerated due to factors such as agriculture and urbanization. Information regarding land use and
cover is essential for various planning and management tasks related to the Earth's surface,
providing crucial environmental data for scientific, resource management, policy purposes, and
diverse human activities.
Accurate understanding of land use and cover is imperative for the development planning
of any area. Consequently, a wide range of professionals, including earth system scientists, land
and water managers, and urban planners, are interested in obtaining data on land use and cover
changes, conversion trends, and other related patterns. The spatial dimensions of land use and
cover support policymakers and scientists in making well-informed decisions, as alterations in
these patterns indicate shifts in economic and social conditions. Monitoring such changes with the
help of Advanced technologies like Remote Sensing and Geographic Information Systems is
crucial for coordinated efforts across different administrative levels. Advanced technologies like
Remote Sensing and Geographic Information Systems
9
Changes in vegetation cover refer to variations in the distribution, composition, and overall
structure of plant communities across different temporal and spatial scales. These changes can
occur natural.
This presentation was provided by Steph Pollock of The American Psychological Association’s Journals Program, and Damita Snow, of The American Society of Civil Engineers (ASCE), for the initial session of NISO's 2024 Training Series "DEIA in the Scholarly Landscape." Session One: 'Setting Expectations: a DEIA Primer,' was held June 6, 2024.
How to Make a Field Mandatory in Odoo 17Celine George
In Odoo, making a field required can be done through both Python code and XML views. When you set the required attribute to True in Python code, it makes the field required across all views where it's used. Conversely, when you set the required attribute in XML views, it makes the field required only in the context of that particular view.
1. B.Sc.III (Sem-VI), Unit-II, Paper-II:Biotechnology
Notes on Southern , Western and Northern Blotting Techniques
Authored by: Dr. Rajendra Chavhan, RMG College Nagbhid
1. SOUTHERN BLOT- DEFINITION, PRINCIPLE, STEPS, RESULTS, APPLICATIONS
Southern Blot Definition
Southern blot is the process of transfer of DNA fragments that are separated by
electrophoresis onto a membrane for immobilization and identification.
Southern blotting has been adopted as a routine procedure for the analysis of
DNA samples for different applications.
The technique was discovered by Edwin Southern, and the technique was
named after him. The technique later gave rise to other techniques
like Western Blotting and Northern Blotting that are based on the same
principle.
The most basic form of the technique is used to determine the size of a DNA
fragment from a complex mixture of genomic DNA.
The technique is also relatively quantitative and can be used to determine the
number of copies of a segment present in a genome.
Southern Blotting can be modified based on the choices of the membrane,
transfer buffer, and method. The most commonly used membrane is the
nitrocellulose membrane, as it is robust and can be reprobed a number of
times.
Similarly, the original protocol of the southern blotting utilizes the use of
radioactive probes; however, other labeling systems utilizing fluorescence
and chemiluminescence.
Southern blotting has been modified in a number of ways to better serve the
application and has been made more complex and efficient.
2. Principle of Southern Blot
The principle of southern blotting is similar to the blotting technique
involving the transfer of biomolecules from a membrane to another for
detection and identification.
The DNA to be analyzed is digested with restriction enzymes and fractionated
by size by the process of agarose gel electrophoresis.
The DNA strands are denatured by alkaline treatment and are transferred to a
nylon or nitrocellulose membrane by the blotting process.
The strands on the membrane are immobilized on the surface by baking or
UV irradiation. The DNA sequences on the membrane can be detected by the
process of hybridization.
Hybridization reactions are specific as the probes used bind to target
fragments consisting of complementary sequences.
The probes used are labeled with different components that can be visualized
by different methods depending on the type of probes used.
Requirements
Equipment
Water Bath
Agarose gel
Power supply
UV radiation
Hybridization Oven
Hybridization Bottles
Trays
3. Film processor
Pipettes
Centrifuge Tubes
Glass Plate
Whatman 3 mm chromatography paper
Nylon membrane/ Nitrocellulose membrane
Syringe
Cellulose acetate membrane
Materials
Restriction Enzymes
Restriction enzymes buffer
Agarose
TBE buffer
DNA loading buffer
Tris base
Sodium chloride
Sodium hydroxide
Sodium citrate
DNA labeling kit
Nucleic acid detection kit
Sodium dodecyl sulfate (SDS)
Polyvinylpyrrolidone
Bovine serum albumin
Formamide
Phenol
Solutions and Buffers
Denaturation buffer: NaOH and NaCl in the ratio 1:6
Neutralization buffer: Tris-HCl and NaCl in the ratio 5:3
SSC: 175.3 g of NaCl and 88.2 g of Sodium citrate to 1L of distilled water.
Detection Buffer: Tris-HCl and NaCl in the ratio 5:1.
Procedure of Southern Blot
a. Restriction digestion of DNA
About 10 µg of the extracted genomic DNA is digested with the appropriate
restriction enzyme in a microcentrifuge tube.
The tube is incubated overnight at 37°C. In some cases, the tubes are heated
in a water bath at 65°C for 20 minutes after the incubation to denature the
restriction enzymes.
To the tubes, 10µl of the DNA sample buffer is added, and the mixture is
poured on agarose gel for electrophoresis.
4. b. Electrophoresis
The percentage and size of the gel are determined based on the size of the
DNA fragments to be separated. The gel is then prepared accordingly.
The electrophoresis buffer is prepared with ethidium bromide and poured into
the tank in a way that is a few millimeters above the gel support.
The gel cast is prepared along with a comb with teeth to form wells that can
hold the sample volume. Once the comb is in place, the gel is slowly poured
into the cast.
Once the gel has set, the comb is removed, and the gel is placed on the tank.
Running buffer is added to the tank to cover the gel.
The samples are prepared by adding loading buffer and carefully pipetted into
the wells.
The tank is connected to the power supply and allowed to run overnight.
c. Denaturation
The gel is removed from the electrophoresis apparatus and placed in a glass
tray with 500 ml denaturation buffer (1.5 M NaCl and 0.5 M NaOH) for 45
minutes at room temperature.
The denaturation buffer is poured off and replaced with a neutralization
buffer. The gel is allowed to soak for 1 hour while slowly rotating on a
platform rotator.
d. Blotting
An oblong sponge that is slightly larger than the gel is placed on a glass dish
which is filled with SSC to leave the soaked sponge about half-submerged in
the buffer.
5. Three pieces of Whatman 3mm paper are cut the same size as the sponge.
These are placed on the sponge and wet with SSC.
The gel is placed on the filter paper and squeezed out to remove bubbles by
rolling a glass pipette over the surface.
A nylon membrane, just large enough to cover the surface of the gel is placed
on top of the gel. The membrane is further flooded with SSC, and few sheets
of filter paper are placed on top of it.
Finally, a glass plate is laid on top of the structure to hold everything in place.
The DNA transfer is allowed to occur overnight.
e. Baking/ Immobilization
The nylon membrane is removed from the blotting structure and attached to
a vacuum or regular oven at 80°C for 2-3 hours.
The DNA strands on the membrane can also be immobilized by exposing the
membrane to ultraviolet radiation.
f. Hybridization
The membrane is exposed to the hybridization probe, which can either be a
DNA fragment or an RNA segment with a specific sequence that detects the
target DNA.
The probe nucleic acid is labeled so that it can be detected by incorporating
radioactivity or tagging the molecules with fluorescent or chromogenic dye.
The conditions during the process are chosen in a way that the probe
hybridizes the target DNA with a complementary sequence on the membrane.
The hybridization is followed by washing with a buffer to remove the probe
that is bound nonspecifically or remain unbound so that only labeled probes
remain bound to the target sequence.
g. Detection
The hybridized regions on the membrane can be detected via autoradiography
by placing the nylon membrane in contact with a photographic film.
The images indicate the position of the hybridized DNA molecules, which
can be used to determine the length of the fragments by comparing them with
the marker DNA molecules of known length.
Similarly, the images also provide information about the number of the
hybridizing fragments and their size.
If a fluorescent or a chromogenic dye is used, these can be visualized on X-
ray film or by the development of color on the membrane.
Result Interpretation of Southern Blot
The results of a Southern blot are observed in the form of bands on the
membrane. The size of the DNA fragments can be determined by comparing
their relative size with the DNA bands of known lengths.
Applications of Southern Blot
1. Southern blotting has many applications in the field of gene discovery,
mapping, evolution, and diagnostic studies.
6. 2. The technique can be used for DNA analysis to detect point mutations and
other structural rearrangements in the DNA sequences.
3. The method also allows the determination of molecular weights of the
restriction fragments, which helps in the analysis of such fragments.
4. Since the technique enables the detection of a particular DNA segment, it can
be used in personal identification via fingerprinting.
5. It can be used in disease diagnosis as well as prenatal diagnosis of genetic
diseases.
2. WESTERN BLOTTING?
Introduction
Western blot is the analytical technique used in molecular biology,
immunogenetics, and other molecular biology to detect specific proteins in a
sample of tissue homogenate or extract. Western blotting is called so as the
procedure is similar to Southern blotting. While Southern blotting is done to
detect DNA, Western blotting is done for the detection of proteins. Western
blotting is also called protein immunoblotting because an antibody is used to
specifically detect its antigen.
Principle of Western blotting
The technique consists of three major processes:
1. Separation of proteins by size (Electrophoresis).
2. Transfer to a solid support (Blotting)
3. Marking target protein using a proper primary and secondary antibody to
visualize (Detection).
Electrophoresis used to separate proteins according to their electrophoretic
mobility which depends on the charge, size of protein molecule, and structure of
7. the proteins. Proteins are moved from within the gel onto a membrane made of
Nitrocellulose (NC) or Polyvinylidene difluoride (PVDF). Without pre-
activation, proteins combine with nitrocellulose membrane based on hydrophobic
interaction (Blotting). For detection of the proteins, primary antibody and
enzyme-conjugated secondary antibody are used. In addition of substrate, a
substrate reacts with the enzyme that is bound to the secondary antibody to
generate colored substance, namely, visible protein bands.
In this technique, a mixture of proteins is separated based on molecular
weight, and thus by type, through gel electrophoresis. These results are then
transferred to a membrane producing a band for each protein. The membrane is
then incubated with labels antibodies specific to the protein of interest. The
unbound antibody is washed off leaving only the bound antibody to the protein
of interest. The bound antibodies are then detected by developing the film. As the
antibodies only bind to the protein of interest, only one band should be visible.
The thickness of the band corresponds to the amount of protein present; thus
doing a standard can indicate the amount of protein present.
Western blotting is usually done on a tissue homogenate or extract. It
utilizes SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel
electrophoresis), a type of gel electrophoresis to first separate various proteins
in a mixture on the basis of their shape and size. The protein bands thus obtained
are transferred onto a nitrocellulose or nylon membrane where they are
“probed” with antibodies specific to the protein to be detected. The antigen-
antibody complexes that form on the band containing the protein recognized by
the antibody can be visualized in a variety of ways.
If the protein of interest is bound by a radioactive antibody, its position on
the blot can be determined by exposing the membrane to a sheet of X-ray film, a
procedure called autoradiography. However, the most generally used detection
procedures employ enzyme-linked antibodies against the protein. After binding
of the enzyme–antibody conjugate, the addition of a chromogenic substrate that
produces a highly colored and insoluble product causes the appearance of a
colored band at the site of the target antigen. The site of the protein of interest can
be determined with a much higher sensitivity if a chemiluminescent compound
along with suitable enhancing agents is used to produce light at the antigen site.
Applications of Western blotting
1. Identification of a specific protein in a complex mixture of proteins. In this
method, known antigens of well-defined molecular weight are separated by
SDS-PAGE and blotted onto nitrocellulose. The separated bands of known
antigens are then probed with the sample suspected of containing antibodies
specific to one or more of these antigens. The reaction of an antibody with a
band is detected by using either a radiolabeled or enzyme-linked secondary
antibody that is specific for the species of the antibodies in the test sample.
2. Estimation of the size of the protein as well as the amount of protein present
in the mixture.
8. 3. It is most widely used as a confirmatory test for the diagnosis of HIV, where
this procedure is used to determine whether the patient has antibodies that
react with one or more viral proteins or not.
4. Demonstration of specific antibodies in the serum for diagnosis of
neurocysticercosis and tubercular meningitis.
4. NORTHERN BLOT- DEFINITION, PRINCIPLE, STEPS, RESULTS, APPLICATIONS
Northern Blot Definition
Northern blot is a technique based on the principle of blotting for the analysis of
specific RNA in a complex mixture.
The technique is a modified version of the Southern Blotting, which was
discovered for the analysis of DNA sequences.
The detection of certain sequences of nucleic acids extracted from different
types of biological samples is essential in molecular biology, which makes
blotting techniques imperative in the field.
The principle is identical to southern blotting except for the probes used for
the detection as northern blotting detects RNA sequences.
This technique provides information about the length of the RNA sequences
and the presence of variations in the sequence.
Even though the technique is primarily focused on the identification of RNA
sequences, it has also been used for the quantification of RNA sequences.
Since the discovery of the technique, several modifications have been made
in the technique for the analysis of mRNAs, pre-mRNAs, and short RNAs.
Northern blotting was employed as the primary technique for the analysis of
RNA fragments for a long time; however, new, more convenient, and cost-
effective techniques like RT-PCR have slowly replaced the technique.
9. Principle of Northern Blot
The principle of the northern blot is the same as all other blotting technique
that is based on the transfer of biomolecules from one membrane to another.
The RNA samples are separated on gels according to their size by gel
electrophoresis. Since RNAs are single-stranded, these can form secondary
structures by intermolecular base pairing. The electrophoretic separation of
the RNA segments is thus performed under denaturing conditions.
The separated RNA fragments are then transferred to a nylon membrane.
Nitrocellulose membrane is not used as RNA doesn’t bind effectively to the
membrane.
The transferred segments are immobilized onto the membrane by fixing
agents. The RNA fragments on the membrane are detected by the addition of
a labeled probe complementary to the RNA sequences present on the
membrane.
The hybridization forms the basis of the detection of RNA as the specificity
of hybridization between the probe, and the RNA allows the accurate
identification of the segments.
Northern blot utilizes size-dependent separation of RNA segments and thus
can be used to determine the sizes of the transcripts.
Requirements
Equipment
Agarose Gel cast
Power Supply
Microwave
Centrifuge
Heating block
UV crosslinker
Hybridization oven
Hybridization vessels
Vials
Forceps
Pipettes
Glass tubes
Materials
Agarose gel
Sodium citrate
Ethylenediaminetetraacetic acid disodium salt dehydrate
NaOH
HCl
Formaldehyde
Glycerol
Ethidium bromide
10. Bromophenol Blue
RNA ladder
MgCl2
NaCl
Polyvinylpyrrolidone
Bovine Serum Albumin
SDS
NaH2PO4
Tris-HCl
Triton-X
DTT
Taq buffer
Taq polymerase
Procedure/Steps of Northern Blot
a. Separation of RNA on a denaturing gel
The RNA gel solution is prepared by adding formaldehyde to the agarose
solution.
The cast is assembled, and the prepared denaturing gel is poured into the cast.
As the gel begins to set, a comb with appropriate teeth is added to form wells.
Once the gel is set, the comb is removed, and the gel is equilibrated with a
running buffer for 30 minutes before running.
15 µg RNA sample is mixed with an equal volume of RNA loading buffer.
Three µg of RNA markers are added in the same volume of RNA loading
buffer.
The samples are incubated at 65°C on a heating block for about 12-15
minutes.
The samples are loaded to the equilibrated gel, and the first row of wells is
filled with RNA markers.
The gel is then run at 125V for about 3 hours.
11. b. Transfer of RNA from gel to the nylon membrane
A nylon membrane is cut that is larger than the size of the denaturing gel, and
a filter paper with the same size as the nylon membrane is also prepared.
Once the electrophoresis process is complete, the RNA gel is removed from
the tank and rinsed with water.
An oblong sponge that is slightly larger than the gel is placed on a glass dish,
and the dish is filled with SSC to a point so as to leave the soaked sponge
about half-submerged in the buffer.
A few pieces of Whatman 3mm papers are placed on top of the sponge and
are wetted with SSC buffer.
The gel is then placed on top of the filter paper and squeezed out to remove
air bubbles by rolling a glass pipette over the surface.
The nylon membrane prepared is wetted with distilled water on an RNase-
free dish for about 5 minutes.
The wetted membrane is placed on the surface of the gel while avoiding any
air bubbles formation.
The surface is further flooded with SSC, and a few more filter papers are
placed on top of the membrane.
12. A glass plate is placed on top of the structure in order to hold everything in
place. The structure is left overnight to obtain an effective transfer.
c. Immobilization
Once the transfer is complete, the gel is removed and rinsed with SSC, and
allowed to dry.
The membrane is placed between two pieces of filter paper and baked in a
vacuum oven at 80°C for 2 hours.
In some cases, the membrane can be wrapped in a UV transparent plastic wrap
and irradiates for an appropriate time on a UV transilluminator.
d. Hybridization
The DNA or RNA probes to be used are to be labeled to a specific activity of
>108 dpm/µg, and unincorporated nucleotides are to be removed.
The membrane carrying the immobilized RNA is wetted with SSC.
The membrane is placed in a hybridization tube with the RNA-side-up, and 1
ml of formaldehyde solution is added.
The tube is placed in the hybridization oven and incubated at 42°C for 3 hours.
If the probe used is double-stranded, it is denatured by heating in a water bath
or incubator for 10 minutes at 100°C.
The desired volume of the probe is pipette into the hybridization tube and
further incubated at 42°C.
The solution is poured off, and the membrane is washed with a wash solution.
The membrane is then observed under autoradiography.