Elution of DNA fragments from agarose gel Practical
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3 Steps are involved binding with silica filter washing through washing buffer elution thruogh low salt elution buffer
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Rflp technology
RFLP is a technique that differentiates organisms by analyzing patterns in DNA fragments produced after digestion with restriction enzymes. If two organisms differ in the distance between restriction sites, the lengths of fragments produced will differ. These patterns can differentiate species and strains. RFLP detection relies on comparing band profiles after digestion and gel electrophoresis to see length polymorphisms, which can then be examined further through hybridization and visualization. RFLPs have applications in forensics, disease detection, and human population genetics.
Polyacrylamide gel electrophoresis
PAGE is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels use as the support matrix.
widely used and has very much importance.
COMPLETE PROCEDURE & USES are described in the slide.
2d Page
2D-PAGE is a technique used to separate complex protein mixtures based on isoelectric point and molecular weight. It involves two sequential steps - isoelectric focusing and SDS-PAGE. In isoelectric focusing, proteins are separated based on their isoelectric point in an immobilized pH gradient. They are then separated by SDS-PAGE based on their molecular weight. The separated proteins can then be visualized through staining and identified through mass spectrometry. While useful for proteomic analysis, 2D-PAGE has limitations such as low reproducibility and dynamic range.
Transfection in animals & plants
Transfection is a technique used to insert foreign nucleic acids like DNA or RNA into cells to alter their properties. There are various biological, chemical, and physical methods to accomplish transfection, either transiently or stably. In stable transfection, the foreign DNA integrates into the cellular genome and is passed to daughter cells, while transient transfection only expresses the DNA for a short time without integration. Genetic engineering techniques are used to transfer genes between organisms, like retrovirus-mediated gene transfer where the retrovirus acts as a vector to deliver transgenes into host cells. Embryonic stem cell-mediated gene transfer involves introducing DNA into stem cells that can integrate randomly or through homologous recombination and be passed to offspring. Liposome
Random Amplified polymorphic DNA. RAPD
RAPD markers are decamer DNA fragments.
RAPD is a type of PCR reaction.
as the name suggest it is a fast method when compared to the traditional PCR medthod.
Southern hybridization
Southern blotting is a technique used to detect specific DNA sequences in a DNA sample. It involves extracting DNA from cells, cutting the DNA into fragments using restriction enzymes, separating the fragments via gel electrophoresis, transferring the DNA fragments to a membrane, and using a labeled probe to detect fragments that are complementary to the probe through hybridization. Southern blotting is useful for identifying mutations, DNA fingerprinting, and detecting DNA in applications like prenatal screening and forensics. While effective for detecting specific DNA sequences, it is a complex, time-consuming, and labor-intensive technique.
Blotting techniques
This document describes three types of blotting techniques - Southern blotting, Northern blotting, and Western blotting. Southern blotting is used to detect DNA fragments separated by agarose gel electrophoresis. Northern blotting detects specific RNA sequences separated by gel electrophoresis. Western blotting identifies proteins separated by SDS-PAGE gel using an antibody probe. The document provides detailed procedures and applications for each type of blotting.
Library screening
This document discusses different types of DNA libraries and methods for screening libraries to identify clones containing genes of interest. It describes genomic and cDNA libraries, noting that genomic libraries contain all DNA fragments from an organism's genome while cDNA libraries contain only coding sequences. The key screening methods discussed are colony/plaque hybridization using radiolabeled probes, expression screening using antibodies, and PCR screening using gene-specific primers.
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Rflp technology
RFLP is a technique that differentiates organisms by analyzing patterns in DNA fragments produced after digestion with restriction enzymes. If two organisms differ in the distance between restriction sites, the lengths of fragments produced will differ. These patterns can differentiate species and strains. RFLP detection relies on comparing band profiles after digestion and gel electrophoresis to see length polymorphisms, which can then be examined further through hybridization and visualization. RFLPs have applications in forensics, disease detection, and human population genetics.
Polyacrylamide gel electrophoresis
PAGE is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels use as the support matrix.
widely used and has very much importance.
COMPLETE PROCEDURE & USES are described in the slide.
2d Page
2D-PAGE is a technique used to separate complex protein mixtures based on isoelectric point and molecular weight. It involves two sequential steps - isoelectric focusing and SDS-PAGE. In isoelectric focusing, proteins are separated based on their isoelectric point in an immobilized pH gradient. They are then separated by SDS-PAGE based on their molecular weight. The separated proteins can then be visualized through staining and identified through mass spectrometry. While useful for proteomic analysis, 2D-PAGE has limitations such as low reproducibility and dynamic range.
Transfection in animals & plants
Transfection is a technique used to insert foreign nucleic acids like DNA or RNA into cells to alter their properties. There are various biological, chemical, and physical methods to accomplish transfection, either transiently or stably. In stable transfection, the foreign DNA integrates into the cellular genome and is passed to daughter cells, while transient transfection only expresses the DNA for a short time without integration. Genetic engineering techniques are used to transfer genes between organisms, like retrovirus-mediated gene transfer where the retrovirus acts as a vector to deliver transgenes into host cells. Embryonic stem cell-mediated gene transfer involves introducing DNA into stem cells that can integrate randomly or through homologous recombination and be passed to offspring. Liposome
Random Amplified polymorphic DNA. RAPD
RAPD markers are decamer DNA fragments.
RAPD is a type of PCR reaction.
as the name suggest it is a fast method when compared to the traditional PCR medthod.
Southern hybridization
Southern blotting is a technique used to detect specific DNA sequences in a DNA sample. It involves extracting DNA from cells, cutting the DNA into fragments using restriction enzymes, separating the fragments via gel electrophoresis, transferring the DNA fragments to a membrane, and using a labeled probe to detect fragments that are complementary to the probe through hybridization. Southern blotting is useful for identifying mutations, DNA fingerprinting, and detecting DNA in applications like prenatal screening and forensics. While effective for detecting specific DNA sequences, it is a complex, time-consuming, and labor-intensive technique.
Blotting techniques
This document describes three types of blotting techniques - Southern blotting, Northern blotting, and Western blotting. Southern blotting is used to detect DNA fragments separated by agarose gel electrophoresis. Northern blotting detects specific RNA sequences separated by gel electrophoresis. Western blotting identifies proteins separated by SDS-PAGE gel using an antibody probe. The document provides detailed procedures and applications for each type of blotting.
Library screening
This document discusses different types of DNA libraries and methods for screening libraries to identify clones containing genes of interest. It describes genomic and cDNA libraries, noting that genomic libraries contain all DNA fragments from an organism's genome while cDNA libraries contain only coding sequences. The key screening methods discussed are colony/plaque hybridization using radiolabeled probes, expression screening using antibodies, and PCR screening using gene-specific primers.
PAGE- Electrophoresis
It is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels used as support media.
Gels are made by free radical-induced polymerization of acrylamide and N,N’-Methylenebisacrylamide.
It is the most widely used technique of electrophoresis.
Rna isolatn
Ribonucleic acid (RNA) can be isolated from plant tissue through several methods for downstream applications. The document describes procedures for RNA isolation using denaturing buffers, phenol-chloroform extraction, and CTAB and TRIzol-based methods. Precautions like maintaining an RNase-free environment and using DEPC-treated materials are important for obtaining high-quality RNA. The procedures involve homogenizing tissues, separating RNA from other cellular components, and precipitating and purifying the isolated RNA.
2 d gel electrophoresis
It will give idea about two dimentional gel electrophoresis. Very Useful PPT. It prepared from so many research papers and books
Screenable and Selectable Markers
Genetic manipulation of plant and animal cells have to be confirmed for further application. One such confirmatory method is the use of stains/dyes which produces fluorescence when the recombination is successful.
Isolation & Purification of DNA
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Labelling of dna
This document discusses nucleic acid probes and their use in hybridization experiments. It notes that probes are short sequences of nucleotides that bind to specific target sequences. The degree of homology between the probe and target determines how stable the hybridization is. Probes can range in size from 10 to over 10,000 nucleotide bases, with most common probes being 14 to 40 bases. Short probes hybridize quickly but have less specificity, while longer probes hybridize more stably. The document then describes different methods for labeling probes, including nick translation, primer extension, RNA polymerase transcription, end-labeling, and direct labeling. It also discusses factors that affect probe specificity and hybridization conditions.
Northern blotting
This presentation is all about Northern blotting technique ..which is a biotechnological process used in cell and molecular biology studies.
GENE KNOCKOUT
A gene knockout is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). However, gene knockout can also refer to the gene that is knocked out or the organism that carries the gene knockout. Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss. Researchers draw inferences from the difference between the knockout organism and normal individuals.
Gene Silencing
This document provides an overview of gene silencing. It begins with definitions of gene silencing and discusses how it differs from gene knockout. The document then covers the short history of gene silencing research from the 1990s onwards. It describes different methods of gene silencing including transcriptional gene silencing and post-transcriptional gene silencing. Specific gene silencing techniques like RNA interference are explained in more detail. The document also includes a case study on gene silencing in petunias and discusses applications of RNAi.
Presentation on marker genes
In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA.
Pyrosequencing
Pyrosequencing is a sequencing method that detects DNA polymerase activity by measuring the release of pyrophosphate using a cascade of enzymatic reactions that generate visible light. It utilizes emulsion PCR to amplify DNA fragments on beads in microreactors. The beads are then loaded into wells and sequenced by sequentially adding nucleotides and detecting light produced upon incorporation using a CCD camera. Key advantages are its accuracy, high throughput of up to 48,000 probes per day, and ease of automation. However, it requires specialized equipment and software.
C value
The document discusses the C-value paradox, which is the lack of relationship between genome size and organism complexity. It provides data on the wide range of genome sizes across different taxonomic groups. Introns and exons are described, with exons comprising the coding sequences and introns being removed from transcripts by splicing. Alternative splicing can generate multiple protein isoforms from a single gene. Repeated sequences, including satellites, minisatellites, microsatellites, transposons, SINEs and LINEs comprise a large portion of eukaryotic genomes.
Colony hybridization technique
Colony hybridization is a technique used to identify bacterial colonies containing a specific gene. It involves transferring bacterial colonies from a master plate onto a nitrocellulose filter to create replicas. The filter is then treated to lyse the bacterial cells and denature the DNA. A radioactive probe is used to hybridize to the target DNA sequence, and autoradiography identifies colonies containing the gene of interest. Colony hybridization allows efficient screening of bacterial colonies to isolate a specific DNA sequence.
2 -D GEL ELECTROPHORESIS
wo-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell and Klose in 1975.
Sts
Sequence tagged sites (STSs) are short DNA sequences that can be used as genetic markers. STSs were introduced in 1989 as a way to map genes along chromosomes using PCR. They serve as landmarks on physical maps of genomes. STSs are mapped by breaking genomes into fragments, replicating the fragments in bacterial cells to create libraries, and using PCR to determine which fragments contain STSs. Different types of STS markers include microsatellites, SCARs, CAPs, and ISSRs, each of which has distinct characteristics and applications in genetic mapping, population studies, and other areas.
Organellar genome
Organellar genomes, such as those found in mitochondria and chloroplasts, can be manipulated. The mitochondria genome is maternally inherited and contains genes that code for proteins involved in respiration. The chloroplast genome is also maternally inherited and contains genes for photosynthesis-related proteins. Methods to transform these genomes include particle bombardment, PEG-mediated transformation, and Agrobacterium-mediated transformation. Manipulating organellar genomes has applications for crop improvement like developing cytoplasmic male sterility.
Protein dna interactions
Protein-DNA interactions can be either specific or non-specific. Specific interactions involve transcription factors that regulate gene expression by binding to DNA motifs through domains like helix-loop-helix, leucine zipper, or zinc finger motifs. Non-specific interactions involve histones that help structure DNA into nucleosomes within chromatin and can be chemically modified through methylation, demethylation, acetylation, and phosphorylation.
Analytical centrifugation
Analytical centrifugation is a technique used to characterize macromolecules based on how they sediment in a centrifugal field. The document discusses the instrumentation, working principle, and two main types of analysis - sedimentation velocity and sedimentation equilibrium. Sedimentation velocity provides information about shape, mass, and size by monitoring the boundary formed over time as particles sediment. Sedimentation equilibrium determines mass composition by analyzing the particle distribution once equilibrium between sedimentation and diffusion is reached. Analytical centrifugation is useful for determining properties like molecular weight, stoichiometry, assembly, and conformation.
DNA footprinting
DNA footprinting is a technique used to identify protein binding regions on DNA. It involves treating DNA with nucleases like DNase I, which will degrade the DNA except for regions bound by proteins. These protected regions, called footprints, can identify transcription factor binding sites that regulate gene expression. The technique was originally developed in 1978 to study the binding specificity of the lac repressor protein, and it provides information on DNA-protein interactions and transcriptional regulation.
Pulse Field Gel Electrophoresis
Pulsed-field gel electrophoresis (PFGE) is a technique used to separate large DNA molecules and generate DNA fingerprints for bacterial isolates. It involves using restriction enzymes to cut bacterial DNA into large fragments, which are then separated in an agarose gel using an electric field that periodically changes direction. PFGE allows discrimination of bacterial strains and is commonly used in epidemiological studies to link clinical infections to environmental or food isolates. While time-consuming, it provides stable, reproducible patterns and has been shown to be more discriminating than other subtyping methods for many bacteria.
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This document describes several methods for isolating and purifying DNA, RNA, and bacteriophages from plant and bacterial cells. For DNA isolation from plant cells, the method involves freezing and grinding plant tissue, lysing the cells with CTAB buffer, purifying the DNA with chloroform, precipitating it with isopropanol, washing it, and eluting the purified DNA. For plasmid and bacteriophage DNA isolation from bacterial cells, several techniques are described that separate DNA based on size or conformation differences, such as alkaline lysis and CsCl gradient centrifugation. RNA isolation methods include organic extraction to separate RNA from other cell components, as well as direct lysis methods.
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The document describes the basic steps for isolating DNA from bacterial cells or plant tissues. There are 5 main steps: 1) disruption of cells to release DNA, 2) inhibition of DNA-degrading enzymes, 3) dissociation of DNA-protein complexes, 4) removal of contaminants, and 5) precipitation of purified DNA. The procedure provided isolates genomic DNA from cauliflower tissue specifically using a spooling method. Key steps include grinding the tissue, lysing cells with detergent-salt buffer, removing proteins and precipitating the DNA with alcohol for collection on a glass rod.
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It is the most widely used technique of electrophoresis.
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Ribonucleic acid (RNA) can be isolated from plant tissue through several methods for downstream applications. The document describes procedures for RNA isolation using denaturing buffers, phenol-chloroform extraction, and CTAB and TRIzol-based methods. Precautions like maintaining an RNase-free environment and using DEPC-treated materials are important for obtaining high-quality RNA. The procedures involve homogenizing tissues, separating RNA from other cellular components, and precipitating and purifying the isolated RNA.
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It will give idea about two dimentional gel electrophoresis. Very Useful PPT. It prepared from so many research papers and books
Screenable and Selectable Markers
Genetic manipulation of plant and animal cells have to be confirmed for further application. One such confirmatory method is the use of stains/dyes which produces fluorescence when the recombination is successful.
Isolation & Purification of DNA
Isolation and Purification of Chromosomal DNA,Plasmid DNA,Bacteriophage DNA used in Recombinant DNA Technology or Biotechnology to produce Recombinant DNA or Desired DNA
Labelling of dna
This document discusses nucleic acid probes and their use in hybridization experiments. It notes that probes are short sequences of nucleotides that bind to specific target sequences. The degree of homology between the probe and target determines how stable the hybridization is. Probes can range in size from 10 to over 10,000 nucleotide bases, with most common probes being 14 to 40 bases. Short probes hybridize quickly but have less specificity, while longer probes hybridize more stably. The document then describes different methods for labeling probes, including nick translation, primer extension, RNA polymerase transcription, end-labeling, and direct labeling. It also discusses factors that affect probe specificity and hybridization conditions.
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This presentation is all about Northern blotting technique ..which is a biotechnological process used in cell and molecular biology studies.
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A gene knockout is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). However, gene knockout can also refer to the gene that is knocked out or the organism that carries the gene knockout. Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss. Researchers draw inferences from the difference between the knockout organism and normal individuals.
Gene Silencing
This document provides an overview of gene silencing. It begins with definitions of gene silencing and discusses how it differs from gene knockout. The document then covers the short history of gene silencing research from the 1990s onwards. It describes different methods of gene silencing including transcriptional gene silencing and post-transcriptional gene silencing. Specific gene silencing techniques like RNA interference are explained in more detail. The document also includes a case study on gene silencing in petunias and discusses applications of RNAi.
Presentation on marker genes
In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA.
Pyrosequencing
Pyrosequencing is a sequencing method that detects DNA polymerase activity by measuring the release of pyrophosphate using a cascade of enzymatic reactions that generate visible light. It utilizes emulsion PCR to amplify DNA fragments on beads in microreactors. The beads are then loaded into wells and sequenced by sequentially adding nucleotides and detecting light produced upon incorporation using a CCD camera. Key advantages are its accuracy, high throughput of up to 48,000 probes per day, and ease of automation. However, it requires specialized equipment and software.
C value
The document discusses the C-value paradox, which is the lack of relationship between genome size and organism complexity. It provides data on the wide range of genome sizes across different taxonomic groups. Introns and exons are described, with exons comprising the coding sequences and introns being removed from transcripts by splicing. Alternative splicing can generate multiple protein isoforms from a single gene. Repeated sequences, including satellites, minisatellites, microsatellites, transposons, SINEs and LINEs comprise a large portion of eukaryotic genomes.
Colony hybridization technique
Colony hybridization is a technique used to identify bacterial colonies containing a specific gene. It involves transferring bacterial colonies from a master plate onto a nitrocellulose filter to create replicas. The filter is then treated to lyse the bacterial cells and denature the DNA. A radioactive probe is used to hybridize to the target DNA sequence, and autoradiography identifies colonies containing the gene of interest. Colony hybridization allows efficient screening of bacterial colonies to isolate a specific DNA sequence.
2 -D GEL ELECTROPHORESIS
wo-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell and Klose in 1975.
Sts
Sequence tagged sites (STSs) are short DNA sequences that can be used as genetic markers. STSs were introduced in 1989 as a way to map genes along chromosomes using PCR. They serve as landmarks on physical maps of genomes. STSs are mapped by breaking genomes into fragments, replicating the fragments in bacterial cells to create libraries, and using PCR to determine which fragments contain STSs. Different types of STS markers include microsatellites, SCARs, CAPs, and ISSRs, each of which has distinct characteristics and applications in genetic mapping, population studies, and other areas.
Organellar genome
Organellar genomes, such as those found in mitochondria and chloroplasts, can be manipulated. The mitochondria genome is maternally inherited and contains genes that code for proteins involved in respiration. The chloroplast genome is also maternally inherited and contains genes for photosynthesis-related proteins. Methods to transform these genomes include particle bombardment, PEG-mediated transformation, and Agrobacterium-mediated transformation. Manipulating organellar genomes has applications for crop improvement like developing cytoplasmic male sterility.
Protein dna interactions
Protein-DNA interactions can be either specific or non-specific. Specific interactions involve transcription factors that regulate gene expression by binding to DNA motifs through domains like helix-loop-helix, leucine zipper, or zinc finger motifs. Non-specific interactions involve histones that help structure DNA into nucleosomes within chromatin and can be chemically modified through methylation, demethylation, acetylation, and phosphorylation.
Analytical centrifugation
Analytical centrifugation is a technique used to characterize macromolecules based on how they sediment in a centrifugal field. The document discusses the instrumentation, working principle, and two main types of analysis - sedimentation velocity and sedimentation equilibrium. Sedimentation velocity provides information about shape, mass, and size by monitoring the boundary formed over time as particles sediment. Sedimentation equilibrium determines mass composition by analyzing the particle distribution once equilibrium between sedimentation and diffusion is reached. Analytical centrifugation is useful for determining properties like molecular weight, stoichiometry, assembly, and conformation.
DNA footprinting
DNA footprinting is a technique used to identify protein binding regions on DNA. It involves treating DNA with nucleases like DNase I, which will degrade the DNA except for regions bound by proteins. These protected regions, called footprints, can identify transcription factor binding sites that regulate gene expression. The technique was originally developed in 1978 to study the binding specificity of the lac repressor protein, and it provides information on DNA-protein interactions and transcriptional regulation.
Pulse Field Gel Electrophoresis
Pulsed-field gel electrophoresis (PFGE) is a technique used to separate large DNA molecules and generate DNA fingerprints for bacterial isolates. It involves using restriction enzymes to cut bacterial DNA into large fragments, which are then separated in an agarose gel using an electric field that periodically changes direction. PFGE allows discrimination of bacterial strains and is commonly used in epidemiological studies to link clinical infections to environmental or food isolates. While time-consuming, it provides stable, reproducible patterns and has been shown to be more discriminating than other subtyping methods for many bacteria.
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The document describes the basic steps for isolating DNA from bacterial cells or plant tissues. There are 5 main steps: 1) disruption of cells to release DNA, 2) inhibition of DNA-degrading enzymes, 3) dissociation of DNA-protein complexes, 4) removal of contaminants, and 5) precipitation of purified DNA. The procedure provided isolates genomic DNA from cauliflower tissue specifically using a spooling method. Key steps include grinding the tissue, lysing cells with detergent-salt buffer, removing proteins and precipitating the DNA with alcohol for collection on a glass rod.
Dna extraction
The document discusses various DNA extraction methods from biological samples. It describes organic extraction, non-organic extraction, Chelex extraction, and FTA paper methods. Organic extraction uses phenol-chloroform to remove proteins and isolate DNA, while non-organic extraction uses high salt concentrations. Chelex extraction uses an ion exchange resin to remove ions that degrade DNA. FTA paper allows samples to be stored at room temperature. The minimum DNA quantities needed for different techniques is also discussed.
RNA isolation
RNA isolation is the process of purifying RNA from biological samples. Three common methods are described:
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2) Filter-based methods use spin baskets and membranes to bind and isolate RNA through centrifugation, providing convenience but risking clogging and retaining gDNA.
3) Magnetic particle methods use paramagnetic beads coated with molecules to bind RNA, which can then be rapidly collected in a magnetic field and resuspended for easier processing than organic extractions. All methods aim to isolate RNA while inhibiting RNases.
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To obtain DNA in a relatively purified form which can be used for further investigations, i.e. PCR, sequencing
Abbas Morovvati
This document provides information about extracting RNA and DNA from biological samples. It discusses the necessary equipment, the purpose of extraction, and the basic steps of the extraction process. For DNA extraction, the main steps involve lysing cells to expose the DNA, removing contaminants, and precipitating the purified DNA. For RNA extraction, similar steps are followed but special precautions must be taken due to RNA's instability. The document also notes common methods for RNA extraction and calculating concentrations of extracted RNA and DNA samples.
DNA_Extraction_Overview
DNA can be extracted from plant cells using a process involving lysis, precipitation, washing, and resuspension. Lysis breaks open the cell walls and membranes to release DNA. Precipitation separates DNA from other cell components using salts and alcohol. Washing removes impurities, and resuspension stores DNA in a stable buffer. While research labs use phenol/chloroform for cleaner DNA, classroom extractions omit this for safety, yielding fragmented but usable DNA seen as smears on gels rather than single bands. Both produce DNA suitable for PCR and other downstream applications.
Gel Purification and Western Blot
This document outlines the basic steps for gel purification of DNA fragments, which includes isolating the bands of interest from an agarose gel, extracting the DNA from those bands, and removing accompanying salts and stain. It describes three common methods for isolating and cleaning the DNA fragment - spin column extraction, dialysis, and the traditional method using phenol/chloroform extraction.
Purification of nucleic acids
Effective disruption of the biological matrix (cell, tissue, environmental or biological sample) to release the nucleic acids. Denaturation of structural proteins associated with the nucleic acids (nucleoproteins) Inactivation of nucleases that will degrade the isolated product (RNase and/or DNase).
Once the genomic DNA is bound to the silica membrane, the nucleic acid is washed with a salt/ethanol solution. These washes remove contaminating proteins, lipopolysaccharides and small RNAs to increase purity while keeping the DNA bound to the silica membrane column.
There are five basic steps of DNA extraction that are consistent across all the possible DNA purification chemistries:
disruption of the cellular structure to create a lysate,
separation of the soluble DNA from cell debris and other insoluble material,
binding the DNA of interest to a purification matrix,
washing proteins and other contaminants away from the matrix and
elution of the DNA.
Rice dna extraction miniprep protocol
DNA extraction is an important step in molecular assays and plays a vital role in obtaining highresolution results in gel-based systems, particularly in the case of cereals with high content of interfering components in the early steps of DNA extraction.This is a rapid miniprep DNA extraction method, optimized for rice, which was achieved via creating some modifications in present DNA extraction methods, especially in first step of breaking down and lyses of cell wall, and the use of cheap and frequent chemicals, found in every lab, in the next steps. The normal quality and quantity was obtained by the method. The PCR based assays also revealed the efficiency of the method.
The advantages of this method are: 1- it is applicable with both dry and fresh samples, 2- no need to large weight samples, 3- no need to liquid nitrogen and 4- easy, rapid and applicable in every laboratory.
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This document provides instructions for extracting genomic DNA from human blood. The objectives are to isolate DNA and analyze it using agarose gel electrophoresis. The extraction process uses EDTA to inhibit DNase, Tris as a buffer, SDS as a detergent to disrupt cell membranes and release DNA, and NaCl and alcohol for precipitation. Blood is collected and red blood cells are lysed using lysis buffer. White blood cells are then lysed and DNA is precipitated using NaCl and ethanol before being collected and analyzed.
Isolation of genomic DNA from Bacterial cell.pptx
The main purpose of these slides is to convey information to the Professors, Lecturers, and Students. These slides contain authentic information about this topic which is mentioned in that.
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Environmental biodegradation of PLA by Biotic and Abiotic factors
PLA is a biodegradable polymer that can degrade through both biotic and abiotic factors in the environment. Biotic degradation occurs through the action of microorganisms like bacteria and fungi that produce enzymes to break down PLA. Specific bacteria identified to degrade PLA include species of Pseudomonas and Streptomyces. Fungal degradation is also possible, with Phanerochaete chrysosporium shown to effectively degrade PLA. Abiotic degradation happens through hydrolysis when water breaks the ester bonds of PLA, which is accelerated at higher temperatures and pH levels.
Energy expenditure and BMR
The document discusses energy expenditure and basal metabolic rate (BMR). It defines energy expenditure as the amount of energy needed for bodily functions like breathing and circulation, while BMR is the minimum energy required for essential physiological processes when at rest. The document outlines several factors that affect BMR, such as age, gender, weight, and thyroid function. Maintaining caloric balance between intake and expenditure through diet and exercise can prevent weight gain.
Agriculture applications of nanobiotechnology
This document discusses the potential applications of nanobiotechnology in agriculture. It begins by introducing how nanoparticles can interact with agricultural hosts and tissues. It then discusses several specific applications, including using nanoparticles for plant disease management and diagnostics, as well as for delivering pesticides, nutrients, and plant hormones. The document also notes potential applications in areas like recycling agricultural waste, soil improvement, water purification, and plant breeding. It acknowledges both the promise and challenges of nanotechnology for modernizing agriculture to address issues like increasing food supply to support population growth amid changing environmental conditions.
Macronutrients and nutrition
Macronutrients provide energy and are essential for growth and maintenance of the body. The document discusses the three main macronutrients - carbohydrates, proteins, and fats. Carbohydrates are divided into simple and complex categories, with simple carbs like sugars providing quick energy and complex carbs like whole grains being more filling and nutritious. Proteins are essential building blocks and energy sources, with animal products providing complete proteins and plant sources providing complementary proteins when combined. Fats serve various functions in the body and are classified based on their structure.
Poly lactic Acid Biodegradation
The document discusses methods to enhance the biodegradation of polylactic acid (PLA). It analyzes modifications to PLA's physical properties and amending the environment with various factors like stimulants. It summarizes that biodegradation of PLA mainly occurs through hydrolysis of ester bonds and is induced by microorganisms like certain actinomycetes, bacteria, and fungi. Key factors like temperature, pH, humidity, and oxygen levels also affect the degradation rate. While PLA is biodegradable, the process is often slow under natural conditions.
Alzhemier's disease and koraskoff syndrome
Alzheimer's disease, Korsakoff's syndrome, and dreaming are compared and contrasted. Alzheimer's disease results from neuronal death and synapse loss, causing memory loss and dementia. Korsakoff's syndrome is caused by thiamine deficiency and can be reversed if treated early. Dreams occur during REM sleep and may help with memory consolidation. Both diseases involve memory loss and neuronal/synaptic changes, while dreaming is a normal process that occurs during sleep and differs in its effects on memory and brain activity.
Nerve cells, Nervous communication & its link to the celllular signalling
The document discusses the structure and function of neurons. It notes that neurons are specialized cells that communicate via electrical and chemical signals. They contain dendrites that receive signals, a cell body, and an axon that transmits signals. At synapses, chemical neurotransmitters transmit signals between neurons or to other cell types. Neurons form circuits that allow for complex coordinated responses. The action potential involves changes in ion channel permeability that propagate electrical signals rapidly along axons. Calcium acts as an important intracellular messenger in neurons and other cell types, often working through the calcium sensor protein calmodulin.
Peptide Hormones and Catecholamines
Peptide hormones and catecholamines allow for rapid responses to environmental changes. They are stored in secretory vesicles and released via exocytosis within seconds or minutes in response to stimulation. This causes short-term effects that are terminated once the hormones are degraded. In contrast, steroid hormones and thyroid hormones are synthesized from cholesterol or thyroglobulin precursors within cells. They diffuse out of cells and circulate in the blood bound to carrier proteins. This allows their effects to last longer, from hours to days, but production and release takes longer than for peptide hormones and catecholamines. The different hormone types thus allow for both rapid short-term responses and longer-term regulatory effects.
Membrane Proteins & its types
Integral & Peripheral membrane proteins
Helical membrane proteins could be single pass /multi pass
porins which are beta sheets ion channels.
membrane lipids & its types
lipids are categorized into 4 major categories.
1) phospholipids
2) glycolipids
3) steroids
4) archeal membrane lipids
Biomembranes (lipids, proteins, carbohydrates)
It includes composition of membranes (fluid mosaic model)
where these membranes are present?
what functions membranes perform?
cell to cell signalling
Cells in multicellular organisms communicate through elaborate signaling networks involving hundreds of signaling molecules. These molecules allow cells to regulate development, growth, and coordinated function. Signaling occurs through paracrine, synaptic, and endocrine mechanisms using molecules like hormones, neurotransmitters, and growth factors. Target cells contain receptors that recognize signaling molecules with high specificity and affinity. While some responses are rapid, others involve long-term changes through regulated synthesis, release, and degradation of signaling compounds.
Protein Folding Mechanism
Folding depends upon sequence of Amino Acids not the Composition. Folding starts with the secondary structure and ends at quaternary structure.
Denaturation occur at secondary, tertiary & quaternary level but not at primary level.
Proetin Tertiary Structure
Tertiary Structure basically of Hydrophobic interactions, (interactions in side chains), hydrogen bonding, salt bridges, Vander Waals interactions.
e.g. Globular proteins & Fibrous Proteins
Restriction digestion
Restriction Enzymes are used to digest DNA at specific Restriction sites(flanking ends) to produce ends that are either cohesive or blunt.
Polymerase Chain Reaction(PCR)
Amplifying DNA through polymerase chain reaction,
3 steps in PCR
Denaturation
Annealing
Extension
More from Sabahat Ali (20)
RECOMBINATION MOLECULAR BIOLOGY PPT UPDATED new.pptx
RECOMBINATION MOLECULAR BIOLOGY PPT UPDATED new.pptx
Good laboratory practices in a pharmaceutical lab 1
Good laboratory practices in a pharmaceutical lab 1
Degradation of PLA at Mesophillic and thermophillic conditions
Degradation of PLA at Mesophillic and thermophillic conditions
Environmental biodegradation of PLA by Biotic and Abiotic factors
Environmental biodegradation of PLA by Biotic and Abiotic factors
Nerve cells, Nervous communication & its link to the celllular signalling
Nerve cells, Nervous communication & its link to the celllular signalling
Recently uploaded
Evidence of Jet Activity from the Secondary Black Hole in the OJ 287 Binary S...
Wereport the study of a huge optical intraday flare on 2021 November 12 at 2 a.m. UT in the blazar OJ287. In the binary black hole model, it is associated with an impact of the secondary black hole on the accretion disk of the primary. Our multifrequency observing campaign was set up to search for such a signature of the impact based on a prediction made 8 yr earlier. The first I-band results of the flare have already been reported by Kishore et al. (2024). Here we combine these data with our monitoring in the R-band. There is a big change in the R–I spectral index by 1.0 ±0.1 between the normal background and the flare, suggesting a new component of radiation. The polarization variation during the rise of the flare suggests the same. The limits on the source size place it most reasonably in the jet of the secondary BH. We then ask why we have not seen this phenomenon before. We show that OJ287 was never before observed with sufficient sensitivity on the night when the flare should have happened according to the binary model. We also study the probability that this flare is just an oversized example of intraday variability using the Krakow data set of intense monitoring between 2015 and 2023. We find that the occurrence of a flare of this size and rapidity is unlikely. In machine-readable Tables 1 and 2, we give the full orbit-linked historical light curve of OJ287 as well as the dense monitoring sample of Krakow.
Immersive Learning That Works: Research Grounding and Paths Forward
We will metaverse into the essence of immersive learning, into its three dimensions and conceptual models. This approach encompasses elements from teaching methodologies to social involvement, through organizational concerns and technologies. Challenging the perception of learning as knowledge transfer, we introduce a 'Uses, Practices & Strategies' model operationalized by the 'Immersive Learning Brain' and ‘Immersion Cube’ frameworks. This approach offers a comprehensive guide through the intricacies of immersive educational experiences and spotlighting research frontiers, along the immersion dimensions of system, narrative, and agency. Our discourse extends to stakeholders beyond the academic sphere, addressing the interests of technologists, instructional designers, and policymakers. We span various contexts, from formal education to organizational transformation to the new horizon of an AI-pervasive society. This keynote aims to unite the iLRN community in a collaborative journey towards a future where immersive learning research and practice coalesce, paving the way for innovative educational research and practice landscapes.
JAMES WEBB STUDY THE MASSIVE BLACK HOLE SEEDS
The pathway(s) to seeding the massive black holes (MBHs) that exist at the heart of galaxies in the present and distant Universe remains an unsolved problem. Here we categorise, describe and quantitatively discuss the formation pathways of both light and heavy seeds. We emphasise that the most recent computational models suggest that rather than a bimodal-like mass spectrum between light and heavy seeds with light at one end and heavy at the other that instead a continuum exists. Light seeds being more ubiquitous and the heavier seeds becoming less and less abundant due the rarer environmental conditions required for their formation. We therefore examine the different mechanisms that give rise to different seed mass spectrums. We show how and why the mechanisms that produce the heaviest seeds are also among the rarest events in the Universe and are hence extremely unlikely to be the seeds for the vast majority of the MBH population. We quantify, within the limits of the current large uncertainties in the seeding processes, the expected number densities of the seed mass spectrum. We argue that light seeds must be at least 103 to 105 times more numerous than heavy seeds to explain the MBH population as a whole. Based on our current understanding of the seed population this makes heavy seeds (Mseed > 103 M⊙) a significantly more likely pathway given that heavy seeds have an abundance pattern than is close to and likely in excess of 10−4 compared to light seeds. Finally, we examine the current state-of-the-art in numerical calculations and recent observations and plot a path forward for near-future advances in both domains.
Gadgets for management of stored product pests_Dr.UPR.pdf
Insectsplayamajorroleinthedeteriorationoffoodgrainscausingbothquantitativeandqualitativelosses
Wellprovedthatnogranariescanbefilledwithgrainswithoutinsectsastheharvestedproducecontainegg(or)larvae(or)pupae(or)adultinsectinthembecauseoffieldcarryoverinfestationwhichcannotbeavoidedindevelopingcountrieslikeIndia
Simpletechnologiesfortimelydetectionofinsectsinthestoredproduceandtherebyplantimelycontrolmeasures
Alternate Wetting and Drying - Climate Smart Agriculture
Alternate Wetting and Drying - Climate Smart AgricultureInternational Food Policy Research Institute- South Asia Office
PPT on Alternate Wetting and Drying presented at the three-day 'Training and Validation Workshop on Modules of Climate Smart Agriculture (CSA) Technologies in South Asia' workshop on April 22, 2024. 8.Isolation of pure cultures and preservation of cultures.pdf
Isolation of pure culture, its various method.
Describing and Interpreting an Immersive Learning Case with the Immersion Cub...
Current descriptions of immersive learning cases are often difficult or impossible to compare. This is due to a myriad of different options on what details to include, which aspects are relevant, and on the descriptive approaches employed. Also, these aspects often combine very specific details with more general guidelines or indicate intents and rationales without clarifying their implementation. In this paper we provide a method to describe immersive learning cases that is structured to enable comparisons, yet flexible enough to allow researchers and practitioners to decide which aspects to include. This method leverages a taxonomy that classifies educational aspects at three levels (uses, practices, and strategies) and then utilizes two frameworks, the Immersive Learning Brain and the Immersion Cube, to enable a structured description and interpretation of immersive learning cases. The method is then demonstrated on a published immersive learning case on training for wind turbine maintenance using virtual reality. Applying the method results in a structured artifact, the Immersive Learning Case Sheet, that tags the case with its proximal uses, practices, and strategies, and refines the free text case description to ensure that matching details are included. This contribution is thus a case description method in support of future comparative research of immersive learning cases. We then discuss how the resulting description and interpretation can be leveraged to change immersion learning cases, by enriching them (considering low-effort changes or additions) or innovating (exploring more challenging avenues of transformation). The method holds significant promise to support better-grounded research in immersive learning.
在线办理(salfor毕业证书)索尔福德大学毕业证毕业完成信一模一样
学校原件一模一样【微信：741003700 】《(salfor毕业证书)索尔福德大学毕业证》【微信：741003700 】学位证，留信认证（真实可查，永久存档）原件一模一样纸张工艺/offer、雅思、外壳等材料/诚信可靠,可直接看成品样本，帮您解决无法毕业带来的各种难题！外壳，原版制作，诚信可靠，可直接看成品样本。行业标杆！精益求精，诚心合作，真诚制作！多年品质 ,按需精细制作，24小时接单,全套进口原装设备。十五年致力于帮助留学生解决难题，包您满意。
本公司拥有海外各大学样板无数，能完美还原。
1:1完美还原海外各大学毕业材料上的工艺：水印，阴影底纹，钢印LOGO烫金烫银，LOGO烫金烫银复合重叠。文字图案浮雕、激光镭射、紫外荧光、温感、复印防伪等防伪工艺。材料咨询办理、认证咨询办理请加学历顾问Q/微741003700
【主营项目】
一.毕业证【q微741003700】成绩单、使馆认证、教育部认证、雅思托福成绩单、学生卡等！
二.真实使馆公证(即留学回国人员证明,不成功不收费)
三.真实教育部学历学位认证（教育部存档！教育部留服网站永久可查）
四.办理各国各大学文凭(一对一专业服务,可全程监控跟踪进度)
如果您处于以下几种情况：
◇在校期间，因各种原因未能顺利毕业……拿不到官方毕业证【q/微741003700】
◇面对父母的压力，希望尽快拿到；
◇不清楚认证流程以及材料该如何准备；
◇回国时间很长，忘记办理；
◇回国马上就要找工作，办给用人单位看；
◇企事业单位必须要求办理的
◇需要报考公务员、购买免税车、落转户口
◇申请留学生创业基金
留信网认证的作用:
1:该专业认证可证明留学生真实身份
2:同时对留学生所学专业登记给予评定
3:国家专业人才认证中心颁发入库证书
4:这个认证书并且可以归档倒地方
5:凡事获得留信网入网的信息将会逐步更新到个人身份内，将在公安局网内查询个人身份证信息后，同步读取人才网入库信息
6:个人职称评审加20分
7:个人信誉贷款加10分
8:在国家人才网主办的国家网络招聘大会中纳入资料，供国家高端企业选择人才
Applied Science: Thermodynamics, Laws & Methodology.pdf
When I was asked to give a companion lecture in support of ‘The Philosophy of Science’ (https://shorturl.at/4pUXz) I decided not to walk through the detail of the many methodologies in order of use. Instead, I chose to employ a long standing, and ongoing, scientific development as an exemplar. And so, I chose the ever evolving story of Thermodynamics as a scientific investigation at its best.
Conducted over a period of >200 years, Thermodynamics R&D, and application, benefitted from the highest levels of professionalism, collaboration, and technical thoroughness. New layers of application, methodology, and practice were made possible by the progressive advance of technology. In turn, this has seen measurement and modelling accuracy continually improved at a micro and macro level.
Perhaps most importantly, Thermodynamics rapidly became a primary tool in the advance of applied science/engineering/technology, spanning micro-tech, to aerospace and cosmology. I can think of no better a story to illustrate the breadth of scientific methodologies and applications at their best.
Direct Seeded Rice - Climate Smart Agriculture
Direct Seeded Rice - Climate Smart AgricultureInternational Food Policy Research Institute- South Asia Office
PPT on Direct Seeded Rice presented at the three-day 'Training and Validation Workshop on Modules of Climate Smart Agriculture (CSA) Technologies in South Asia' workshop on April 22, 2024.
Pests of Storage_Identification_Dr.UPR.pdf
InIndia-post-harvestlosses-unscientificstorage,insects,rodents,micro-organismsetc.,accountforabout10percentoftotalfoodgrains
Graininfestation
Directdamage
Indirectly
•theexuviae,skin,deadinsects
•theirexcretawhichmakefoodunfitforhumanconsumption
About600speciesofinsectshavebeenassociatedwithstoredgrainproducts
100speciesofinsectpestsofstoredproductscauseeconomiclosses
The debris of the ‘last major merger’ is dynamically young
The Milky Way’s (MW) inner stellar halo contains an [Fe/H]-rich component with highly eccentric orbits, often referred to as the
‘last major merger.’ Hypotheses for the origin of this component include Gaia-Sausage/Enceladus (GSE), where the progenitor
collided with the MW proto-disc 8–11 Gyr ago, and the Virgo Radial Merger (VRM), where the progenitor collided with the
MW disc within the last 3 Gyr. These two scenarios make different predictions about observable structure in local phase space,
because the morphology of debris depends on how long it has had to phase mix. The recently identified phase-space folds in Gaia
DR3 have positive caustic velocities, making them fundamentally different than the phase-mixed chevrons found in simulations
at late times. Roughly 20 per cent of the stars in the prograde local stellar halo are associated with the observed caustics. Based
on a simple phase-mixing model, the observed number of caustics are consistent with a merger that occurred 1–2 Gyr ago.
We also compare the observed phase-space distribution to FIRE-2 Latte simulations of GSE-like mergers, using a quantitative
measurement of phase mixing (2D causticality). The observed local phase-space distribution best matches the simulated data
1–2 Gyr after collision, and certainly not later than 3 Gyr. This is further evidence that the progenitor of the ‘last major merger’
did not collide with the MW proto-disc at early times, as is thought for the GSE, but instead collided with the MW disc within
the last few Gyr, consistent with the body of work surrounding the VRM.
The binding of cosmological structures by massless topological defects
Assuming spherical symmetry and weak field, it is shown that if one solves the Poisson equation or the Einstein field
equations sourced by a topological defect, i.e. a singularity of a very specific form, the result is a localized gravitational
field capable of driving flat rotation (i.e. Keplerian circular orbits at a constant speed for all radii) of test masses on a thin
spherical shell without any underlying mass. Moreover, a large-scale structure which exploits this solution by assembling
concentrically a number of such topological defects can establish a flat stellar or galactic rotation curve, and can also deflect
light in the same manner as an equipotential (isothermal) sphere. Thus, the need for dark matter or modified gravity theory is
mitigated, at least in part.
ESA/ACT Science Coffee: Diego Blas - Gravitational wave detection with orbita...
ESA/ACT Science Coffee: Diego Blas - Gravitational wave detection with orbita...Advanced-Concepts-Team
Presentation in the Science Coffee of the Advanced Concepts Team of the European Space Agency on the 07.06.2024.
Speaker: Diego Blas (IFAE/ICREA)
Title: Gravitational wave detection with orbital motion of Moon and artificial
Abstract:
In this talk I will describe some recent ideas to find gravitational waves from supermassive black holes or of primordial origin by studying their secular effect on the orbital motion of the Moon or satellites that are laser ranged.MICROBIAL INTERACTION PPT/ MICROBIAL INTERACTION AND THEIR TYPES // PLANT MIC...
MICROBIAL INTERACTION PPT/ MICROBIAL INTERACTION AND THEIR TYPES // PLANT MIC...ABHISHEK SONI NIMT INSTITUTE OF MEDICAL AND PARAMEDCIAL SCIENCES , GOVT PG COLLEGE NOIDA
Microbial interaction
Microorganisms interacts with each other and can be physically associated with another organisms in a variety of ways.
One organism can be located on the surface of another organism as an ectobiont or located within another organism as endobiont.
Microbial interaction may be positive such as mutualism, proto-cooperation, commensalism or may be negative such as parasitism, predation or competition
Types of microbial interaction
Positive interaction: mutualism, proto-cooperation, commensalism
Negative interaction: Ammensalism (antagonism), parasitism, predation, competition
I. Mutualism:
It is defined as the relationship in which each organism in interaction gets benefits from association. It is an obligatory relationship in which mutualist and host are metabolically dependent on each other.
Mutualistic relationship is very specific where one member of association cannot be replaced by another species.
Mutualism require close physical contact between interacting organisms.
Relationship of mutualism allows organisms to exist in habitat that could not occupied by either species alone.
Mutualistic relationship between organisms allows them to act as a single organism.
Examples of mutualism:
i. Lichens:
Lichens are excellent example of mutualism.
They are the association of specific fungi and certain genus of algae. In lichen, fungal partner is called mycobiont and algal partner is called
II. Syntrophism:
It is an association in which the growth of one organism either depends on or improved by the substrate provided by another organism.
In syntrophism both organism in association gets benefits.
Compound A
Utilized by population 1
Compound B
Utilized by population 2
Compound C
utilized by both Population 1+2
Products
In this theoretical example of syntrophism, population 1 is able to utilize and metabolize compound A, forming compound B but cannot metabolize beyond compound B without co-operation of population 2. Population 2is unable to utilize compound A but it can metabolize compound B forming compound C. Then both population 1 and 2 are able to carry out metabolic reaction which leads to formation of end product that neither population could produce alone.
Examples of syntrophism:
i. Methanogenic ecosystem in sludge digester
Methane produced by methanogenic bacteria depends upon interspecies hydrogen transfer by other fermentative bacteria.
Anaerobic fermentative bacteria generate CO2 and H2 utilizing carbohydrates which is then utilized by methanogenic bacteria (Methanobacter) to produce methane.
ii. Lactobacillus arobinosus and Enterococcus faecalis:
In the minimal media, Lactobacillus arobinosus and Enterococcus faecalis are able to grow together but not alone.
The synergistic relationship between E. faecalis and L. arobinosus occurs in which E. faecalis require folic acid
Sexuality - Issues, Attitude and Behaviour - Applied Social Psychology - Psyc...
A proprietary approach developed by bringing together the best of learning theories from Psychology, design principles from the world of visualization, and pedagogical methods from over a decade of training experience, that enables you to: Learn better, faster!
Anti-Universe And Emergent Gravity and the Dark Universe
Recent theoretical progress indicates that spacetime and gravity emerge together from the entanglement structure of an underlying microscopic theory. These ideas are best understood in Anti-de Sitter space, where they rely on the area law for entanglement entropy. The extension to de Sitter space requires taking into account the entropy and temperature associated with the cosmological horizon. Using insights from string theory, black hole physics and quantum information theory we argue that the positive dark energy leads to a thermal volume law contribution to the entropy that overtakes the area law precisely at the cosmological horizon. Due to the competition between area and volume law entanglement the microscopic de Sitter states do not thermalise at sub-Hubble scales: they exhibit memory effects in the form of an entropy displacement caused by matter. The emergent laws of gravity contain an additional ‘dark’ gravitational force describing the ‘elastic’ response due to the entropy displacement. We derive an estimate of the strength of this extra force in terms of the baryonic mass, Newton’s constant and the Hubble acceleration scale a0 = cH0, and provide evidence for the fact that this additional ‘dark gravity force’ explains the observed phenomena in galaxies and clusters currently attributed to dark matter.
Recently uploaded (20)
Evidence of Jet Activity from the Secondary Black Hole in the OJ 287 Binary S...
Evidence of Jet Activity from the Secondary Black Hole in the OJ 287 Binary S...
Immersive Learning That Works: Research Grounding and Paths Forward
Immersive Learning That Works: Research Grounding and Paths Forward
Gadgets for management of stored product pests_Dr.UPR.pdf
Gadgets for management of stored product pests_Dr.UPR.pdf
Alternate Wetting and Drying - Climate Smart Agriculture
Alternate Wetting and Drying - Climate Smart Agriculture
8.Isolation of pure cultures and preservation of cultures.pdf
8.Isolation of pure cultures and preservation of cultures.pdf
Juaristi, Jon. - El canon espanol. El legado de la cultura española a la civi...
Juaristi, Jon. - El canon espanol. El legado de la cultura española a la civi...
Describing and Interpreting an Immersive Learning Case with the Immersion Cub...
Describing and Interpreting an Immersive Learning Case with the Immersion Cub...
Applied Science: Thermodynamics, Laws & Methodology.pdf
Applied Science: Thermodynamics, Laws & Methodology.pdf
The debris of the ‘last major merger’ is dynamically young
The debris of the ‘last major merger’ is dynamically young
The binding of cosmological structures by massless topological defects
The binding of cosmological structures by massless topological defects
ESA/ACT Science Coffee: Diego Blas - Gravitational wave detection with orbita...
ESA/ACT Science Coffee: Diego Blas - Gravitational wave detection with orbita...
MICROBIAL INTERACTION PPT/ MICROBIAL INTERACTION AND THEIR TYPES // PLANT MIC...
MICROBIAL INTERACTION PPT/ MICROBIAL INTERACTION AND THEIR TYPES // PLANT MIC...
Sexuality - Issues, Attitude and Behaviour - Applied Social Psychology - Psyc...
Sexuality - Issues, Attitude and Behaviour - Applied Social Psychology - Psyc...
Anti-Universe And Emergent Gravity and the Dark Universe
Anti-Universe And Emergent Gravity and the Dark Universe
Elution of DNA fragments from agarose gel Practical
- 1. Practical#10 26-dec-2018 Elution of DNA fragments from agarose gel INTRODUCTION: After isolating plasmids it may contain some chromosomal DNA contamination it will interrupt the further processingof cloning. So it is betterto recover the plasmid DNA by eluting it from agarose gels (extraction). Gel-Elution is a standard procedure performed to recover desired DNA fragments from agarose gels after electrophoretic separation. After dissolving the gel fragment and running it through a specialized filter, Advantages of gel filtration: this procedure yields DNA freed from impurities such as salts, free nucleotides and enzymes, suitable for downstream applications. Principle: The basic principlebehind DNA recovery from agarose gel involves a sequence of bind, wash, and elute steps. Once the gel is in solubilizing buffer, it is applied onto a “spin column,” which, upon centrifugation, allows DNA
- 2. molecules to selectively bind to a silica-filter while the impurities flow through into a collection tube.DNA is able to bind to silica thanks to a high salt concentration in the gel solubilizationbuffer. This buffer is believed to disrupt the hydration structure around the filter and create a cationsalt bridge between the strong negative charges on the filter and negative charges on the DNA. Residual impurities are removed by washing with ethanol. Water or low salt buffer is added to the column and will “elute,” or free, the DNA from it, presumably by disrupting the cation bridge. The DNA is now purified from the gel. REQUIREMENTS: Elution buffer(10 mM tris HCl pH 8.5) Scalpel blade UV transilluminator Agarose Micro pipettes Micro pipette tips Dry bath incubator Microfuge tubes Centrifuge Cyclomixer
- 3. 70% Ethanol 95% Ethanol TE buffer( 10 mM Tris HCL,EDTA) -20o C freezer -70o C freezer PROCEDURE: 1.Gel Electrophoresis: The first step in the gel purification procedure involves casting the agarose gel and performing electrophoresis of the DNA samples. Once the gel-run is complete, desired DNA fragments are visualized against UV light and fragments are selected after comparing against a molecular weight standard. If the gel is unstained, the band location can be approximately determined based on a comparison to the DNA ladder. 2.Cutting of Bands: Whilecuttingthegel with a razorblade,onemust take care to recover as much DNA as possible with as little agarose as possible.When handling ethidium bromide stained gels
- 4. and working in front of UV light, gloves and protective eyewear should be used. After cutting the desired DNA from the gel, dispose of the gel and running buffer properly, in compliance with institutional safety protocols.Once isolated, the piece of gel is placed in a microfuge tube and weighed on a balance. Using the approximation that 100 mg of gel occupies100 l, a volume of solublilization buffer that is 4X the gel weight is added to the gel piece. After being placed in buffer, the gel piece is incubated at around 50 ◦ C to melt the agarose On Dry bath incubator.. 3.Once melted, the solubilized gel is added onto a spin column and thesolutionis centrifuged, which will cause all of the DNA and other particulates to stick to the filter. 4.The boundDNA is washed by adding70% ethanol to the filter, followed by centrifugation, which will remove residual impurities from the filter. Flow through is discarded, and this washing step is then generally repeated up to three times. 5. The empty filter is spun again to remove residual ethanol, and the silica filter is allowed to dry at room temperature. Water or elution buffer is added to the filter,
- 5. and with another round of centrifugation, purified DNA is collected in the bottom of the tube.Suspend the pellet in 20µl of TE. 6. If confirmation of DNA is required , run (1µl) of it on gel. The recovered DNA can be now used for further process of cloning otherwise can stored in -20o C freezer.