Polyacrylamide gel electrophoresis (PAGE) is a method used to separate macromolecules like proteins or nucleic acids based on their size and charge. It works by applying an electric current to move the charged molecules through a polyacrylamide gel matrix. Smaller molecules move faster through the gel's pores than larger ones. SDS-PAGE specifically uses sodium dodecyl sulfate detergent to denature proteins and impart a uniform negative charge, allowing separation based primarily on size. The document discusses the principles, procedures, applications and different types of PAGE in detail.
SDS-PAGE is a technique used to separate proteins by molecular weight. Proteins are denatured and given a negative charge by SDS detergent before running through a polyacrylamide gel matrix by electrophoresis. Smaller proteins migrate faster through the gel, allowing separation by size. After electrophoresis, proteins bands can be visualized using stains like Coomassie blue or silver stain to analyze characteristics like molecular weight, purity, and subunit composition.
The document discusses different expression vectors and systems used for recombinant protein expression. It describes key elements required for an expression vector including an origin of replication, selective marker, promoter, multiple cloning site, and terminator. It provides details on commonly used expression systems in E. coli such as the lac, tac, lambda PL, and T7 promoters. It also summarizes protein expression in yeast using the galactose-inducible GAL promoter system.
Gel electrophoresis is a standard lab procedure for separating DNA fragments by size using an electrical current to move negatively charged DNA through an agarose gel matrix. Shorter DNA fragments migrate faster than longer fragments, allowing DNA size to be estimated by comparing to a DNA ladder of known sizes. Key aspects of the technique include using agarose to create a porous gel, a buffer like TAE or TBE to conduct electricity and facilitate DNA movement, and ethidium bromide dye to visualize DNA bands under UV light. The method exploits the fact that DNA migration rate depends on size, with smaller fragments traveling farther than larger ones when subjected to an electric field.
This document describes the blue-white screening technique for identifying recombinant bacteria. Blue-white screening relies on the activity of β-galactosidase, an enzyme in E. coli that cleaves lactose. Plasmid vectors used in cloning carry a fragment of the lacZ gene. When the plasmid inserts foreign DNA at the multiple cloning site, it disrupts lacZ and prevents complementation, so the bacteria cannot metabolize lactose and appear white on an indicator plate. If no DNA inserts or inserts elsewhere, complementation occurs and bacteria appear blue. The technique allows rapid identification of bacteria that took up recombinant plasmid.
1) SDS-PAGE is a technique that separates proteins by molecular weight through gel electrophoresis.
2) Proteins are treated with SDS and β-mercaptoethanol to denature them before loading onto a discontinuous polyacrylamide gel with stacking and separating sections.
3) As proteins migrate through the gel under an electric current, their movement depends on size, with smaller proteins traveling farther, allowing separation by molecular weight.
Agarose gel electrophoresis is a technique used to separate DNA fragments by size. DNA samples are loaded into wells in an agarose gel and an electric current is applied, causing the fragments to migrate through the gel at rates dependent on their size. Smaller fragments travel farther than larger fragments, allowing DNA samples to be separated into bands of distinct sizes that can be visualized after staining.
This document discusses various techniques for gene transfer, including natural methods like conjugation, transformation, and transduction, as well artificial methods like microinjection, biolistics, calcium phosphate and liposome mediated transfer, and electroporation. It provides details on how each method works, such as how conjugation involves transfer of DNA between bacteria via sex pili, and how electroporation uses electrical pulses to create pores in cell membranes to allow DNA entry. The document also summarizes screening and applications of transgenic techniques.
Polyacrylamide gel electrophoresis (PAGE) is a method used to separate macromolecules like proteins or nucleic acids based on their size and charge. It works by applying an electric current to move the charged molecules through a polyacrylamide gel matrix. Smaller molecules move faster through the gel's pores than larger ones. SDS-PAGE specifically uses sodium dodecyl sulfate detergent to denature proteins and impart a uniform negative charge, allowing separation based primarily on size. The document discusses the principles, procedures, applications and different types of PAGE in detail.
SDS-PAGE is a technique used to separate proteins by molecular weight. Proteins are denatured and given a negative charge by SDS detergent before running through a polyacrylamide gel matrix by electrophoresis. Smaller proteins migrate faster through the gel, allowing separation by size. After electrophoresis, proteins bands can be visualized using stains like Coomassie blue or silver stain to analyze characteristics like molecular weight, purity, and subunit composition.
The document discusses different expression vectors and systems used for recombinant protein expression. It describes key elements required for an expression vector including an origin of replication, selective marker, promoter, multiple cloning site, and terminator. It provides details on commonly used expression systems in E. coli such as the lac, tac, lambda PL, and T7 promoters. It also summarizes protein expression in yeast using the galactose-inducible GAL promoter system.
Gel electrophoresis is a standard lab procedure for separating DNA fragments by size using an electrical current to move negatively charged DNA through an agarose gel matrix. Shorter DNA fragments migrate faster than longer fragments, allowing DNA size to be estimated by comparing to a DNA ladder of known sizes. Key aspects of the technique include using agarose to create a porous gel, a buffer like TAE or TBE to conduct electricity and facilitate DNA movement, and ethidium bromide dye to visualize DNA bands under UV light. The method exploits the fact that DNA migration rate depends on size, with smaller fragments traveling farther than larger ones when subjected to an electric field.
This document describes the blue-white screening technique for identifying recombinant bacteria. Blue-white screening relies on the activity of β-galactosidase, an enzyme in E. coli that cleaves lactose. Plasmid vectors used in cloning carry a fragment of the lacZ gene. When the plasmid inserts foreign DNA at the multiple cloning site, it disrupts lacZ and prevents complementation, so the bacteria cannot metabolize lactose and appear white on an indicator plate. If no DNA inserts or inserts elsewhere, complementation occurs and bacteria appear blue. The technique allows rapid identification of bacteria that took up recombinant plasmid.
1) SDS-PAGE is a technique that separates proteins by molecular weight through gel electrophoresis.
2) Proteins are treated with SDS and β-mercaptoethanol to denature them before loading onto a discontinuous polyacrylamide gel with stacking and separating sections.
3) As proteins migrate through the gel under an electric current, their movement depends on size, with smaller proteins traveling farther, allowing separation by molecular weight.
Agarose gel electrophoresis is a technique used to separate DNA fragments by size. DNA samples are loaded into wells in an agarose gel and an electric current is applied, causing the fragments to migrate through the gel at rates dependent on their size. Smaller fragments travel farther than larger fragments, allowing DNA samples to be separated into bands of distinct sizes that can be visualized after staining.
This document discusses various techniques for gene transfer, including natural methods like conjugation, transformation, and transduction, as well artificial methods like microinjection, biolistics, calcium phosphate and liposome mediated transfer, and electroporation. It provides details on how each method works, such as how conjugation involves transfer of DNA between bacteria via sex pili, and how electroporation uses electrical pulses to create pores in cell membranes to allow DNA entry. The document also summarizes screening and applications of transgenic techniques.
pBluescript is an example of a combination between plasmids and phages (phagemids).
Phagemids represent a hybrid type of class of vectors that serve to produce single-stranded DNA.
Arabinose Operon is a self-regulatory sequence of genes used by material to metabolize a five-carbon sugar called arabinose when there is a deficiency of glucose in the environment.
Gene transfer technologies can be used to treat diseases by inserting therapeutic genes into cells. There are viral and non-viral methods of gene transfer. Viral methods use viruses like retroviruses, adenoviruses, and adeno-associated viruses to efficiently deliver genes. Non-viral methods include mechanical techniques like electroporation, microinjection, and biolistics (gene gun), as well as chemical methods like liposomes, calcium phosphate, and polyethylene glycol. Each method has advantages and limitations for different applications in research and potential gene therapy.
Agarose gel electrophoresis is used to separate DNA fragments by size. DNA has a negative charge and will migrate toward the positive electrode in an electrical field. Shorter DNA fragments move faster through an agarose gel than longer fragments. Samples are run alongside a DNA ladder to determine the size of unknown DNA fragments. After electrophoresis, gels are stained with ethidium bromide and visualized under UV light to see DNA band patterns. This allows analysis of results such as identifying the presence of specific genes.
The western blot is a technique used to detect specific proteins in a sample. It involves separating proteins by size using gel electrophoresis, transferring them to a membrane, and using antibodies to detect the target protein. The key steps are sample preparation, gel electrophoresis, blotting, blocking, antibody probing, and detection. Western blotting allows researchers to identify proteins from complex mixtures and is widely used in molecular biology and medical diagnosis, such as detecting HIV, HBV, and HSV infections.
The document discusses various gene transfer techniques, including direct and indirect methods. Direct methods involve physical techniques like electroporation, particle bombardment, and microinjection which directly transfer DNA into cells. Chemical methods also directly transfer DNA using liposomes, DEAE dextran, or calcium phosphate precipitation. Indirect methods involve vector-mediated transfer, including Agrobacterium-mediated transfer where bacteria insert DNA into plant cells, and virus-mediated transfer using viruses like tobacco mosaic virus or cauliflower mosaic virus.
1. Gene regulation in prokaryotes and eukaryotes involves control at the levels of transcription and translation.
2. In prokaryotes, genes are often organized into operons and regulated through inducible and repressible operons controlled by regulatory proteins binding to operator and promoter sites. The lac and trp operons are examples of inducible and repressible operons, respectively.
3. In eukaryotes, gene expression is controlled through chromatin structure, transcriptional initiation, transcript processing and modification, transport, stability, and small RNA-mediated pathways. This allows for complex tissue-specific and developmental control of gene expression.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
pUC vectors are plasmids derived from pBR322 that have a higher copy number of 500-600 copies per cell. They contain an ampicillin resistance gene for selection, as well as the lacZ' gene containing multiple cloning sites. When a gene of interest is inserted, it disrupts the lacZ' gene, allowing for blue-white screening on media containing IPTG and X-gal to identify recombinant colonies that appear white instead of blue. pUC vectors offer advantages over pBR322 such as high copy number and easy selection, though they cannot accommodate inserts larger than 15kb.
Microinjection is a gene transfer technique where DNA is directly injected into cells using a fine glass micropipette. It is highly efficient at the individual cell level and was originally used for transfecting hard-to-transfect cells. The procedure involves holding a cell using one pipette while another pipette is used to inject DNA into the cell's cytoplasm or nucleus. It allows for stable transfection efficiencies of around 20% and is used to generate transgenic animals by injecting DNA into oocytes, eggs or embryos. However, it is time-consuming and can only be done for a small number of cells.
To modifying the structure of a specific gene.
Gene targeting vector introduced into the cell.
Vector modifies the normal chromosomal gene through homologous recombination.
Useful in treating some human genetic disorders – Hemophilia, Duchenne Muscular Dystrophy.
Treating human diseases by genetic approaches – Gene Therapy.
Gene Therapy – Replacing the defective gene by normal copy of the gene.
Expressed sequence tag/EST is a short partial sequence, typically 200-400 bp long, of a complimentary DNA/Cdna.
EST is a short sub-sequence of a cDNA sequence.
Used to identify gene transcripts, and are instrumental in gene discovery and in gene-sequence determination.
Approximately 74.2 million ESTs are available in public databases.
EST results from one-short sequencing of a cloned cDNA.
Low-quality fragments.
Length is approximately 500 to 800 nucleotides.
Agarose gel electrophoresis is a technique used to separate DNA fragments by size. DNA has a negative charge and will migrate toward the positive electrode in an agarose gel, allowing DNA fragments of different sizes to be separated. Key aspects of agarose gel electrophoresis include using an agarose gel made from seaweed extract, loading DNA samples into the gel wells, running a current through the gel to separate the DNA by size, and staining the gel with a dye like ethidium bromide to visualize the DNA fragments under UV light.
This document summarizes the process of agarose gel electrophoresis. Agarose gel is prepared by combining agarose powder with a buffer solution to establish pH and conductivity. Samples are loaded into wells in the gel and an electric current is applied, causing DNA fragments to migrate through the gel at rates corresponding to their size. Agarose gel electrophoresis is used to separate and analyze DNA fragments for applications such as estimating DNA size, analyzing polymerase chain reaction products, and extracting DNA fragments for further purification.
Site-directed mutagenesis is a technique used to introduce specific changes to the DNA sequence of a gene by altering the nucleotide sequence. It allows researchers to study the impact of mutations by changing individual bases, deleting bases, or inserting new bases. There are different methods of site-directed mutagenesis including oligonucleotide-based methods and PCR-based methods. Site-directed mutagenesis has applications in research, production of desired proteins, and development of engineered proteins for commercial uses like detergents.
The MAP kinase pathway is a three-kinase signalling module that transmits signals from growth factor receptors to the nucleus. When Ras is activated by growth factors, it recruits and activates Raf, the first kinase in the cascade. Raf then activates MEK, which activates ERK. Active ERK translocates to the nucleus and phosphorylates transcription factors, influencing processes like cell proliferation. The three-kinase design allows for signal amplification and transmission from the membrane to the nucleus, while regulatory mechanisms maintain specificity.
This document describes SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), a technique used to separate proteins by their molecular weight. SDS denatures proteins and gives them a uniform charge. This allows proteins to be separated solely by size as they migrate through a polyacrylamide gel under an electric field. Smaller proteins migrate faster. The document outlines the SDS-PAGE method and preparation of samples, gels, buffers and staining for visualization and analysis of separated proteins. Applications include determining protein purity, molecular weight and peptide mapping.
This document provides information on gel electrophoresis techniques. It defines electrophoresis as a method to separate macromolecules like DNA, RNA, and proteins based on size and charge using an electrical current applied through a gel. It describes the principles, types of gels used (agarose or polyacrylamide), sample preparation, running procedures, and staining steps for separating DNA, RNA, and proteins. Protocols for agarose gel electrophoresis of DNA, formaldehyde gel electrophoresis of RNA, and SDS-PAGE gel electrophoresis of proteins are outlined in detail.
Gel electrophoresis is a method used to separate DNA, RNA, and proteins based on size and charge by pushing the molecules through a gel with an electrical current. Smaller molecules move faster and further than larger ones. There are two main types of gels used: agarose gels for larger molecules like DNA and RNA, and polyacrylamide gels for smaller molecules like proteins. The document provides detailed protocols for running gel electrophoresis with DNA, RNA, and the differences between the types of gels and their applications in research and clinical settings.
pBluescript is an example of a combination between plasmids and phages (phagemids).
Phagemids represent a hybrid type of class of vectors that serve to produce single-stranded DNA.
Arabinose Operon is a self-regulatory sequence of genes used by material to metabolize a five-carbon sugar called arabinose when there is a deficiency of glucose in the environment.
Gene transfer technologies can be used to treat diseases by inserting therapeutic genes into cells. There are viral and non-viral methods of gene transfer. Viral methods use viruses like retroviruses, adenoviruses, and adeno-associated viruses to efficiently deliver genes. Non-viral methods include mechanical techniques like electroporation, microinjection, and biolistics (gene gun), as well as chemical methods like liposomes, calcium phosphate, and polyethylene glycol. Each method has advantages and limitations for different applications in research and potential gene therapy.
Agarose gel electrophoresis is used to separate DNA fragments by size. DNA has a negative charge and will migrate toward the positive electrode in an electrical field. Shorter DNA fragments move faster through an agarose gel than longer fragments. Samples are run alongside a DNA ladder to determine the size of unknown DNA fragments. After electrophoresis, gels are stained with ethidium bromide and visualized under UV light to see DNA band patterns. This allows analysis of results such as identifying the presence of specific genes.
The western blot is a technique used to detect specific proteins in a sample. It involves separating proteins by size using gel electrophoresis, transferring them to a membrane, and using antibodies to detect the target protein. The key steps are sample preparation, gel electrophoresis, blotting, blocking, antibody probing, and detection. Western blotting allows researchers to identify proteins from complex mixtures and is widely used in molecular biology and medical diagnosis, such as detecting HIV, HBV, and HSV infections.
The document discusses various gene transfer techniques, including direct and indirect methods. Direct methods involve physical techniques like electroporation, particle bombardment, and microinjection which directly transfer DNA into cells. Chemical methods also directly transfer DNA using liposomes, DEAE dextran, or calcium phosphate precipitation. Indirect methods involve vector-mediated transfer, including Agrobacterium-mediated transfer where bacteria insert DNA into plant cells, and virus-mediated transfer using viruses like tobacco mosaic virus or cauliflower mosaic virus.
1. Gene regulation in prokaryotes and eukaryotes involves control at the levels of transcription and translation.
2. In prokaryotes, genes are often organized into operons and regulated through inducible and repressible operons controlled by regulatory proteins binding to operator and promoter sites. The lac and trp operons are examples of inducible and repressible operons, respectively.
3. In eukaryotes, gene expression is controlled through chromatin structure, transcriptional initiation, transcript processing and modification, transport, stability, and small RNA-mediated pathways. This allows for complex tissue-specific and developmental control of gene expression.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
pUC vectors are plasmids derived from pBR322 that have a higher copy number of 500-600 copies per cell. They contain an ampicillin resistance gene for selection, as well as the lacZ' gene containing multiple cloning sites. When a gene of interest is inserted, it disrupts the lacZ' gene, allowing for blue-white screening on media containing IPTG and X-gal to identify recombinant colonies that appear white instead of blue. pUC vectors offer advantages over pBR322 such as high copy number and easy selection, though they cannot accommodate inserts larger than 15kb.
Microinjection is a gene transfer technique where DNA is directly injected into cells using a fine glass micropipette. It is highly efficient at the individual cell level and was originally used for transfecting hard-to-transfect cells. The procedure involves holding a cell using one pipette while another pipette is used to inject DNA into the cell's cytoplasm or nucleus. It allows for stable transfection efficiencies of around 20% and is used to generate transgenic animals by injecting DNA into oocytes, eggs or embryos. However, it is time-consuming and can only be done for a small number of cells.
To modifying the structure of a specific gene.
Gene targeting vector introduced into the cell.
Vector modifies the normal chromosomal gene through homologous recombination.
Useful in treating some human genetic disorders – Hemophilia, Duchenne Muscular Dystrophy.
Treating human diseases by genetic approaches – Gene Therapy.
Gene Therapy – Replacing the defective gene by normal copy of the gene.
Expressed sequence tag/EST is a short partial sequence, typically 200-400 bp long, of a complimentary DNA/Cdna.
EST is a short sub-sequence of a cDNA sequence.
Used to identify gene transcripts, and are instrumental in gene discovery and in gene-sequence determination.
Approximately 74.2 million ESTs are available in public databases.
EST results from one-short sequencing of a cloned cDNA.
Low-quality fragments.
Length is approximately 500 to 800 nucleotides.
Agarose gel electrophoresis is a technique used to separate DNA fragments by size. DNA has a negative charge and will migrate toward the positive electrode in an agarose gel, allowing DNA fragments of different sizes to be separated. Key aspects of agarose gel electrophoresis include using an agarose gel made from seaweed extract, loading DNA samples into the gel wells, running a current through the gel to separate the DNA by size, and staining the gel with a dye like ethidium bromide to visualize the DNA fragments under UV light.
This document summarizes the process of agarose gel electrophoresis. Agarose gel is prepared by combining agarose powder with a buffer solution to establish pH and conductivity. Samples are loaded into wells in the gel and an electric current is applied, causing DNA fragments to migrate through the gel at rates corresponding to their size. Agarose gel electrophoresis is used to separate and analyze DNA fragments for applications such as estimating DNA size, analyzing polymerase chain reaction products, and extracting DNA fragments for further purification.
Site-directed mutagenesis is a technique used to introduce specific changes to the DNA sequence of a gene by altering the nucleotide sequence. It allows researchers to study the impact of mutations by changing individual bases, deleting bases, or inserting new bases. There are different methods of site-directed mutagenesis including oligonucleotide-based methods and PCR-based methods. Site-directed mutagenesis has applications in research, production of desired proteins, and development of engineered proteins for commercial uses like detergents.
The MAP kinase pathway is a three-kinase signalling module that transmits signals from growth factor receptors to the nucleus. When Ras is activated by growth factors, it recruits and activates Raf, the first kinase in the cascade. Raf then activates MEK, which activates ERK. Active ERK translocates to the nucleus and phosphorylates transcription factors, influencing processes like cell proliferation. The three-kinase design allows for signal amplification and transmission from the membrane to the nucleus, while regulatory mechanisms maintain specificity.
This document describes SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), a technique used to separate proteins by their molecular weight. SDS denatures proteins and gives them a uniform charge. This allows proteins to be separated solely by size as they migrate through a polyacrylamide gel under an electric field. Smaller proteins migrate faster. The document outlines the SDS-PAGE method and preparation of samples, gels, buffers and staining for visualization and analysis of separated proteins. Applications include determining protein purity, molecular weight and peptide mapping.
This document provides information on gel electrophoresis techniques. It defines electrophoresis as a method to separate macromolecules like DNA, RNA, and proteins based on size and charge using an electrical current applied through a gel. It describes the principles, types of gels used (agarose or polyacrylamide), sample preparation, running procedures, and staining steps for separating DNA, RNA, and proteins. Protocols for agarose gel electrophoresis of DNA, formaldehyde gel electrophoresis of RNA, and SDS-PAGE gel electrophoresis of proteins are outlined in detail.
Gel electrophoresis is a method used to separate DNA, RNA, and proteins based on size and charge by pushing the molecules through a gel with an electrical current. Smaller molecules move faster and further than larger ones. There are two main types of gels used: agarose gels for larger molecules like DNA and RNA, and polyacrylamide gels for smaller molecules like proteins. The document provides detailed protocols for running gel electrophoresis with DNA, RNA, and the differences between the types of gels and their applications in research and clinical settings.
Agarose gel electrophoresis is a technique used to separate DNA fragments by size using an electrical current applied to an agarose gel. DNA samples are loaded into wells in the gel along with a DNA ladder standard. As the current is applied, smaller DNA fragments migrate farther through the pores of the gel than larger fragments. After running the gel, DNA bands can be visualized by staining with a dye and examining under UV light. This allows determination of whether PCR was successful in amplifying DNA fragments and their approximate size by comparison to the standard ladder.
Gel electrophoresis separates molecules like DNA and proteins based on their size and charge. Agarose gel electrophoresis is commonly used to analyze DNA fragments. Samples are loaded into wells in an agarose gel submerged in buffer solution. When an electric current is applied, negatively charged DNA migrates toward the positive electrode at rates inversely proportional to its size. Staining the gel with a fluorescent dye like ethidium bromide allows visualization of DNA bands under ultraviolet light. This reveals the presence and size of DNA fragments like PCR products.
Gel electrophoresis separates molecules like DNA and proteins based on their size and charge. Agarose gel electrophoresis is commonly used to analyze DNA fragments. Samples are loaded into wells in an agarose gel submerged in buffer solution. When an electric current is applied, negatively charged DNA migrates toward the positive electrode at rates inversely proportional to its size. Staining the gel with ethidium bromide allows visualization of DNA bands under ultraviolet light. This technique can determine the presence and size of DNA fragments like PCR products.
Gel electrophoresis is a technique used to separate DNA fragments by size. An agarose gel is prepared and DNA samples mixed with dye are loaded into wells. When a current is applied, DNA migrates toward the positive electrode based on size, with smaller fragments traveling farther. After running the gel, DNA bands can be visualized under UV light after staining with ethidium bromide or an alternative dye. The document provides details on preparing an agarose gel, running electrophoresis, and analyzing results to determine if samples contain target DNA sequences like those indicating the presence of Wolbachia bacteria.
Electro- = flow of electricity,
-phoresis = to carry across
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely-used techniques in biochemistry and molecular biology
Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field.
Methods & Types
Biol2 Lecture 2 Dna Isolation And Agarose GelEricT1
The document discusses DNA isolation and agarose gel electrophoresis. It describes how DNA can be extracted from various biological sources and purified. The process involves lysing cells, removing proteins, and separating DNA from other molecules. Agarose gel electrophoresis is then used to separate DNA fragments by size, as smaller fragments move more quickly through the gel under an electric field. The document outlines the basic steps for preparing an agarose gel, loading samples mixed with dye, running the gel, and visualizing DNA bands under UV light.
Gel electrophoresis is a technique used to separate DNA fragments by size. An agarose gel is cast and DNA samples mixed with a tracking dye are loaded into wells. When a voltage is applied, DNA migrates through the gel at a rate inversely proportional to its size. Smaller fragments move faster. After running the gel, DNA bands can be visualized by staining with ethidium bromide and viewing under UV light or alternative stains viewed without UV. This allows determination of presence and size of DNA fragments such as PCR products.
Gel electrophoresis is a technique used to separate DNA fragments by size. An agarose gel is cast and DNA samples mixed with a tracking dye are loaded into wells. When a voltage is applied, DNA migrates through the gel at a rate inversely proportional to its size. Smaller fragments move faster. After running the gel, DNA bands can be visualized by staining with ethidium bromide and viewing under UV light or with alternative stains viewed without UV. This allows determination of presence and size of DNA fragments such as PCR products.
Gel electrophoresis is a technique used to separate DNA fragments by size. An agarose gel is cast and placed in an electrophoresis chamber filled with buffer solution. DNA samples mixed with dye are loaded into wells in the gel. An electric current is applied, causing negatively charged DNA fragments to migrate through the agarose gel at rates inversely proportional to their size. Smaller fragments move faster through the gel than larger fragments. After running the gel, DNA bands can be visualized by staining with a fluorescent dye like ethidium bromide and viewing under ultraviolet light. This allows determination of presence and size of DNA fragments in samples.
Gel electrophoresis is a technique used to separate DNA, RNA, and proteins based on their size and charge. During gel electrophoresis, a current is applied to move the molecules through a gel, with smaller molecules moving faster through the pores in the gel. This allows researchers to determine the size of unknown molecules by comparing their migration to molecules of known sizes. Key aspects of gel electrophoresis include preparing an agarose or polyacrylamide gel, loading samples and a ladder, running the current, and visualizing the separated molecules using dyes like ethidium bromide under ultraviolet light.
NUCLEIC ACID EXTRACTION, PURIFICATION ON AGAROSE AND POLYACRYLAMIDE GEL AND PCREmmanuel Nestory Kayuni
The document provides information about DNA and RNA extraction techniques from animal and plant cells. It discusses extracting nucleic acids using kits with varying costs and protocols for extracting DNA from animal tissue and plants. It also summarizes analyzing extracted nucleic acids through electrophoresis on agarose and polyacrylamide gels and using polymerase chain reaction (PCR) for applications such as DNA sequencing, forensics, and population genetics.
Gel electrophoresis is a method to separate molecules like DNA, RNA, or proteins based on their size and charge. It works by placing the molecules in an agarose or polyacrylamide gel and applying an electric current, causing the molecules to migrate through the gel at different rates depending on their size and charge. Smaller and more highly charged molecules move faster. This allows separation of molecules that differ in size by as little as a single nucleotide or amino acid. Common applications include analyzing DNA fragments after enzymatic digestion or PCR, separating proteins by molecular weight, and determining purity of samples.
Electrophoresis is a technique used to separate molecules like proteins and nucleic acids based on their size and charge. It works by applying an electric current to move the molecules through a gel or liquid matrix. There are different types of gels like agarose and polyacrylamide that can be used depending on the size of molecules being separated. Agarose gels are commonly used to separate DNA fragments of different lengths. The process involves casting an agarose gel, loading samples mixed with a dye, running a current through the gel, and visualizing the separated DNA bands.
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.
The webinar may also give some examples on how nonprofits can best leverage Walmart Business+.
The event will cover the following::
Walmart Business + (https://business.walmart.com/plus) is a new shopping experience for nonprofits, schools, and local business customers that connects an exclusive online shopping experience to stores. Benefits include free delivery and shipping, a 'Spend Analytics” feature, special discounts, deals and tax-exempt shopping.
Special TechSoup offer for a free 180 days membership, and up to $150 in discounts on eligible orders.
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.
Temple of Asclepius in Thrace. Excavation resultsKrassimira Luka
The temple and the sanctuary around were dedicated to Asklepios Zmidrenus. This name has been known since 1875 when an inscription dedicated to him was discovered in Rome. The inscription is dated in 227 AD and was left by soldiers originating from the city of Philippopolis (modern Plovdiv).
Level 3 NCEA - NZ: A Nation In the Making 1872 - 1900 SML.pptHenry Hollis
The History of NZ 1870-1900.
Making of a Nation.
From the NZ Wars to Liberals,
Richard Seddon, George Grey,
Social Laboratory, New Zealand,
Confiscations, Kotahitanga, Kingitanga, Parliament, Suffrage, Repudiation, Economic Change, Agriculture, Gold Mining, Timber, Flax, Sheep, Dairying,
The chapter Lifelines of National Economy in Class 10 Geography focuses on the various modes of transportation and communication that play a vital role in the economic development of a country. These lifelines are crucial for the movement of goods, services, and people, thereby connecting different regions and promoting economic activities.
A Visual Guide to 1 Samuel | A Tale of Two HeartsSteve Thomason
These slides walk through the story of 1 Samuel. Samuel is the last judge of Israel. The people reject God and want a king. Saul is anointed as the first king, but he is not a good king. David, the shepherd boy is anointed and Saul is envious of him. David shows honor while Saul continues to self destruct.
Beyond Degrees - Empowering the Workforce in the Context of Skills-First.pptxEduSkills OECD
Iván Bornacelly, Policy Analyst at the OECD Centre for Skills, OECD, presents at the webinar 'Tackling job market gaps with a skills-first approach' on 12 June 2024
Beyond Degrees - Empowering the Workforce in the Context of Skills-First.pptx
DNA agarose gel electrophoresis
1. Molecular biology
DNA AGAROSE GEL ELECTROPHORESIS
Prepared by E. NSENGIYUMVA, BSc
Lab technician
University of Rwanda
College of Medicine and Health
Biomedical Laboratory Sciences
2. INTRODUCTION
Electrophoresis is a technique of separating molecules basing on
their electrical charges, where positively and negatively charged
molecules migrate to cathode (negative) and anode (positive)
poles respectively.
Molecules with the same charges, are further separated
according to their size where larger molecules migrate slower
than small ones.
3. INTRODUCTION(Cont.)
Agarose gel electrophoresis is a routinely used method for separating
DNA or RNA. Nucleic acid molecules are size separated by the aid of
electric field where negatively charged molecules migrate toward anode
pole.
The agarose gel electrophoresis is the most used and effective way of
separating DNA fragments of varying sizes ranging from 100 bp to 25 Kb.
4. Introduction (CONT;)
Agarose gel consists of microscopic pores that act as a molecular sieve
which separates molecules based upon charge, size, and shape.
These characteristics together with buffer conditions, gel concentration
and voltage affect the mobility of molecules in gels.
5. INTRODUCTION(Cont.
Application of DNA agarose gel electrophoresis
DNA fragment size estimation
DNA purification
Genetic fingerprinting
PCR product analysis
6. PRINCIPLE
DNA sample is loaded into pre-cast wells in the agarose gel and a current applied. The phosphate
backbone of the DNA (and RNA) molecules is negatively charged, therefore when placed in an
electric field, DNA fragments will migrate to the positively charged anode.
Because DNA has a uniform mass/charge ratio. DNA molecules are separated by size; the smaller
molecules migrate faster than large molecules
7. Materials and reagents
Materials
Gel casting tray
Gel combs
Electrophoresis chamber
Electrophoresis power supply
Tank cover
Electrical leads
UV transilluminator
Chemical Balance
Erlenmeyer Flask
Oven or heater
Personal protective equipments
Timer
Reagents
50x TAE Buffer
Agarose gel powder
DNA Ladder
Ethidium Bromide or SBR safe
Gel loading dye
Distilled water
8.
9. 1X TAE buffer preparation
1x TAE Buffer is used in agarose gel preparation and for running gel electrophoresis
Prepared from 50x TAE Buffer
Add 20 mL of 50x TAE stock solution to a 1 L Duran bottle.
Add 980 mL of distilled water.
Mix the solution by shaking.
10. Agarose gel preparation
Percent Agarose Gel (W/V) DNA Size Resolution (kb=1000)
0.5% 1 kb to 30 kb
0.7% 800 bp to 12 kb
1.0% 500 bp to 10 kb
1.2% 400 bp to 7 kb
1.5% 200 bp to 3 kb
2.0% 50 bp to 2 kb
Agarose gel is prepared depending on your DNA size
11. 1% Gel preparation
Measure out 25 mL of 1X TAE using a graduated cylinder
Pour into a 100 mL Erlenmeyer flask.
Measure 0.25 g of agarose and add to flask to produce a ~ 1.0% w/v agarose gel.
Heat the flask using a microwave to melt the agarose. Swirl and mix the agarose.
Let cool at 600C for 3 minutes on the bench. Add 2.0 µL (1/10000) of Ethidium
Bromide (EtBr) [Carcinogenic, PPE must be worn) or SYBR Safe and swirl.
Pour onto assembled gel tray with comb.
Use a pipette tip to move any air bubbles to the side or the bottom of the tray.
Agarose will solidify after a few minutes and turn translucent, glossy.
12. Sample loading
Place tray with solidified gel into the gel box, and gently remove the comb
from the gel.
Pour TAE buffer into the gel box, the gel must be covered with buffer.
Carefully add 1 µL of loading dye to each ~ 10 µL DNA sample.
Carefully pipette 7 µL of 1kb ladder into the first lane of the gel.
Load 15 µL each of the samples into separate wells by pipetting, as described
above.
13. Running the gel
Place the lid of the gel box properly (black with black, and red with red).
Run the gel at 100 Volts for about 45 minutes or until the blue dye has run about 1/2 to 2/3
across the gel.
Turn off the current and remove leads.
14. Gel documentation
Remove the lid of the gel box.
Remove gel from the gel box. Drain off excess buffer from the
surface of the gel. Place the gel tray on paper towels to absorb any
extra running buffer.
Remove the gel from the gel tray and put in the gel
documentation machine
Expose the gel to UV light. This is most commonly done using a
gel documentation system. DNA bands should show up as orange
fluorescent bands. Take a picture of the gel and store on flash disk
or in computer for gel fragment size observation.
Properly dispose of the gel after gel documentation or DNA
purification.
16. References
Lee, P
. Y. et al. (2012) ‘Agarose gel electrophoresis for the separation of DNA
fragments’, Journal of Visualized Experiments, (62), pp. 1–5. doi: 10.3791/3923.
Magdeldin, S. (2012) Gel Electrophoresis - Principles and Basics, Gel
Electrophoresis - Principles and Basics. doi: 10.5772/2205.