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.
Agro-bacterium-mediated transformation is a common method for inserting genes into dicotyledonous plants using the soil bacterium Agro-bacterium. The method involves using Agro-bacterium containing a T-DNA vector to transfer genes of interest into plant cells, followed by regeneration of transformed plants on selection media. Protocols are provided for transforming tobacco and chrysanthemum explants using Agro-bacterium, including co-cultivation, selection, regeneration of shoots, rooting of shoots, and verification of transformation. Transformed tobacco and chrysanthemum plants were successfully recovered using this method.
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.
This work is done in IIT-M (Indian Institute of Technology- Madras) with help of Indian Academy of Science during June 2011-Oct 2011 under Dr Karunagaran Devarajan sir
This document describes culture methods for cultivating various protozoan parasites. It discusses the purposes of culturing parasites, including for diagnostic, research, and teaching purposes. It provides examples of parasite species that can be cultured, such as Entamoeba histolytica, Giardia lamblia, and Plasmodium spp. The document outlines different types of culture media, including xenic, polyxenic, monoxenic, and axenic cultures. It also describes specific culture media and methods used for cultivating intestinal protozoa like amoebae, as well as haematozoan parasites including Leishmania and trypanosomes.
This document provides instructions for using an ELISA kit to detect the mycotoxin zearalenone in cereal crops and animal feeds. It begins with an introduction to zearalenone and its health effects. It then describes the intended use, principle, reagents, materials, precautions, extraction procedure, and assay procedure for the zearalenone ELISA kit. The kit is designed to quantitatively detect zearalenone in samples through a competitive enzyme immunoassay.
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.
Agro-bacterium-mediated transformation is a common method for inserting genes into dicotyledonous plants using the soil bacterium Agro-bacterium. The method involves using Agro-bacterium containing a T-DNA vector to transfer genes of interest into plant cells, followed by regeneration of transformed plants on selection media. Protocols are provided for transforming tobacco and chrysanthemum explants using Agro-bacterium, including co-cultivation, selection, regeneration of shoots, rooting of shoots, and verification of transformation. Transformed tobacco and chrysanthemum plants were successfully recovered using this method.
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.
This work is done in IIT-M (Indian Institute of Technology- Madras) with help of Indian Academy of Science during June 2011-Oct 2011 under Dr Karunagaran Devarajan sir
This document describes culture methods for cultivating various protozoan parasites. It discusses the purposes of culturing parasites, including for diagnostic, research, and teaching purposes. It provides examples of parasite species that can be cultured, such as Entamoeba histolytica, Giardia lamblia, and Plasmodium spp. The document outlines different types of culture media, including xenic, polyxenic, monoxenic, and axenic cultures. It also describes specific culture media and methods used for cultivating intestinal protozoa like amoebae, as well as haematozoan parasites including Leishmania and trypanosomes.
This document provides instructions for using an ELISA kit to detect the mycotoxin zearalenone in cereal crops and animal feeds. It begins with an introduction to zearalenone and its health effects. It then describes the intended use, principle, reagents, materials, precautions, extraction procedure, and assay procedure for the zearalenone ELISA kit. The kit is designed to quantitatively detect zearalenone in samples through a competitive enzyme immunoassay.
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.
The general procedure for plant tissue culture involves sterilizing glassware and tools, preparing and sterilizing explant tissue samples, producing callus growth from the explants on nutrient media, proliferating the callus through subculture, and establishing suspension cultures. Key steps include surface sterilizing explants using chemicals like sodium hypochlorite, transferring sterilized explants to growth media, incubating to produce initial callus, subculturing callus periodically to fresh media, and creating suspension cultures by transferring callus to liquid shaking media.
The document discusses plant protoplast isolation, purification, and culturing. Some key points:
- Protoplasts are plant cells that have had their cell walls removed, leaving just the plasma membrane. They allow for plant cell fusion and regeneration.
- Protoplasts are typically isolated from plant tissues like leaves using enzymatic digestion with cellulase and pectinase. This yields more protoplasts than mechanical methods.
- Isolated protoplasts are purified by centrifugation and washing to remove cell debris. They are then cultured in liquid or solid nutrient media and tested for viability before regeneration.
Protoplasm fusion involves removing the cell wall from plant or bacterial cells through enzymatic digestion to create protoplasts. Protoplasts can be fused through spontaneous fusion during isolation or induced fusion using various methods like mechanical manipulation, chemical fusogens, or electricity. Successful fusion results in a hybrid cell containing genetic material from both parent cells. Protoplast fusion allows for combining genes from sexually incompatible species and is useful for plant breeding and genetic engineering applications.
Transformation of tomato using CRISPER- CAS9 construct to enhance the shelf l...ShreyasGowda87
This document summarizes a student project to transform tomato plants using CRISPR-Cas9 to enhance shelf life. The objectives are to standardize tomato hypocotyl regeneration and optimize genetic transformation using a CRISPR construct targeting two tomato pectate lyase genes. The methodology involves germinating tomato seeds, taking hypocotyl explants, co-cultivating with Agrobacterium containing the CRISPR construct, regenerating plants on selection media, and rooting regenerants to obtain transgenic plants with increased shelf life. Preliminary results showed regeneration of putative transgenic shoots from several batches of explants.
This document outlines the steps for conducting an ethnobotanical study and screening collected plants. It involves selecting an unexplored study area, collecting ethnobotanical knowledge from local people, classifying and preserving collected plant species, screening 10 plants for further analysis using qualitative and quantitative methods, and extracting, amplifying, and detecting DNA from samples. The goal is to systematically evaluate traditional uses of plants and characterize the biochemical and molecular basis of these uses.
This document discusses cytogenetics and chromosome analysis techniques. It begins with an introduction to human chromosomes and chromosomal abnormalities. It then describes various types of chromosomal mutations and abnormalities that can be detected through karyotyping and fluorescence in situ hybridization (FISH). The document provides detailed procedures for chromosome sample preparation from bone marrow and blood cultures, as well as staining and analysis techniques like Giemsa staining and G-banding. The importance of chromosomal studies for diagnosing conditions like Turner syndrome and Klinefelter syndrome is also highlighted.
This document describes procedures for plant regeneration through callus culture using tobacco plants. It involves initiating callus cultures from tobacco explant tissue, observing the growth pattern of callus cultures over multiple phases, and inducing shoot and root organogenesis from the callus to regenerate tobacco plants. The objectives are to initiate and maintain callus cultures from tobacco explants, create cell suspension cultures, and induce organ development from callus to regenerate whole plants. Standard procedures are provided for callus initiation, maintenance, measurement of growth curves, and regeneration of shoots and roots. Results showed high rates of callus formation and organogenesis from explants and callus on optimal media formulations.
This document provides a detailed tutorial on the procedure for culturing human melanocytes. Key steps include:
1) Isolating epidermis from skin specimens using dispase enzyme solution overnight at 4°C.
2) Dispersing epidermal cells using trypsin and mechanically dissociating into a single cell suspension.
3) Seeding cells in TPA-free growth medium and incubating without disturbance for 48-72 hours.
4) Maintaining cultures by changing medium twice weekly and passaging confluent cultures using trypsin.
Methods for cryopreserving and thawing melanocytes are also described. Morphology and growth characteristics of cultured melanocytes are provided.
This document provides instructions for performing an enzyme-linked immunosorbent assay (ELISA) to quantitatively detect total aflatoxins (B1, B2, G1, and G2) in grains, nuts, and other commodities. Aflatoxins are toxic metabolites produced by certain molds that can contaminate foods and animal feeds. The ELISA uses an aflatoxin-specific antibody to competitively bind sample aflatoxins or HRP-conjugated aflatoxin. The intensity of the color reaction indicates the aflatoxin concentration in samples, which can be quantified by comparison to standard curves. Validation studies showed the assay can reliably detect aflatoxins from 1-20 p
This document provides instructions for using an ochratoxin A assay kit. It describes ochratoxin A as a mycotoxin produced by molds that is toxic and carcinogenic in humans and animals. The kit is intended to quantitatively detect ochratoxin A in grains, coffee, cocoa, spices and other foods. It works by competitively binding ochratoxin A from standards and samples to an antibody, and then detecting the amount of bound conjugate using a colorimetric reaction. Detailed procedures are provided for extracting ochratoxin A from different sample types and running the assay.
This document discusses the process of tissue processing in histopathology. It describes the key steps which include:
1. Tissue collection, fixation to prevent decay, and gross examination.
2. Processing which involves dehydration, clearing, infiltration with paraffin wax, and embedding tissues in paraffin blocks for sectioning.
3. Sectioning tissues with a microtome and staining, typically with hematoxylin and eosin, for microscopic examination.
This presentation gives all the basic necessary details about cell culture and karyotyping in cell lines . It describes the important methods like subculture,confluency,cell count,passaging and karyotyping in cell culture.It also describes about BSL.It also gives a brief idea about types of cell culture.This presentation also includes the requirements in the cell culture lab
This presentation deals tissue processing in histopathology, the detailed of presentation given blow:
Histology, study the organization of tissues at all levels, from the whole organ down to the molecular components of cells that are found in most multicellular plants and animals.
Animal tissues are classified as epithelium, with closely spaced cells and very little intercellular space; connective tissue, with large amounts of intercellular material; muscle, specialized for contraction; and nerve, specialized for conduction of electrical impulses. Blood is also sometimes considered a separate tissue type.
Plants are composed of relatively undifferentiated tissue known as meristematic tissue; storage tissue or parenchyma; vascular tissue; photosynthetic tissue or chlorenchyma and support tissue or sclerenchyma and collenchyma.
nettle propsal presentation power point Afaf final copy.pptxAfafAbuhilal
This document presents a literature review and methodology for a study on the extraction of bioactive compounds from stinging nettle (Urtica dioica). Key points:
1. Stinging nettle is a common weed with stinging hairs that contains various nutrients, minerals, vitamins, proteins, and polyphenols like ursolic acid and quercetin that have medical benefits.
2. The study will extract bioactive compounds using solvent extraction methods like Soxhlet extraction and determine total phenolic content and antioxidant activity. It will also examine antimicrobial effects.
3. A literature review found that ethanol extraction via Soxhlet had the highest yields in other studies. Parameters like solvent, temperature
This document describes the microbial limit test, which includes tests to quantify and qualify microorganisms in samples. It involves estimating total viable counts of bacteria and fungi, and detecting specific pathogens. The test is based on culturing samples on various media to support or inhibit growth of target microbes. Methods like membrane filtration, spread plating, and serial dilution are used to quantify microbes, while selective media help identify pathogens like E. coli, S. aureus, P. aeruginosa, and Salmonella. Detailed procedures are provided for quantification, enrichment, and identification of microorganisms in samples.
Human liver microsomes & rat liver microsomesgaurav sharma
Human and rat liver microsomes are subcellular fractions containing cytochrome P450 enzymes and other drug-metabolizing enzymes. They are commonly used in vitro to study drug metabolism and interactions. Human liver microsomes are obtained from human liver tissue through differential centrifugation and contain enzymes for phase I and phase II drug metabolism. They are useful for identifying drug metabolites, evaluating interspecies differences, predicting in vivo clearance, and studying interindividual variability in drug metabolism. Rat liver microsomes were also discussed as an experimental model. Incubation methods and analytical techniques like HPLC were described for evaluating metabolism using liver microsomes.
The extraction of DNA involves three main steps that are cell lysis, protein separation, and DNA purification. Cell lysis is usually performed by incubation of cell in buffer containing detergent and protease. Cellular proteins are salted out or phase separated using organic solvents. Finally DNA is isolated and purified either by alcohol precipitation or adsorption with silica and elution.
1) The document summarizes different types of cell viability assays, including dye exclusion assays, colorimetric assays, fluorometric assays, and luminometric assays.
2) Specific assays discussed in more detail include trypan blue exclusion assay, MTT assay, SRB assay, and 5-CFDA-AM fluorometric assay.
3) The assays are described in terms of their principles, protocols, calculation methods, and applications in measuring cell viability and screening drug responses.
Nguyen Thi Nhi completed the following works in the third week of her master's program:
1) Learned techniques for MIN6 cell culture including making media and using an autoclave.
2) Studied cell culture and immunoassay techniques in theory.
3) Specifically learned how to make cell culture media including DMEM, FBS, penicillin/streptomycin, and beta-mercaptoethanol and the purpose of each component. She also learned proper operation of the JSR autoclave for sterilization.
This document discusses bead-based separation techniques for isolating biomolecules like DNA. It explains that magnetic beads containing iron oxide particles can selectively bind to target molecules like DNA under certain buffer and salt conditions. The DNA is then separated and purified from other contaminants by applying a magnetic field to pull the beads and attached DNA out of solution while leaving behind other molecules. The DNA is later released from the beads in low salt buffer for downstream applications. The technique allows for rapid separation and purification of nucleic acids without centrifugation or filtration.
The document discusses the process of micropropagation through adventitious shoot proliferation using African violet as an example. There are four main stages: 1) initiation of sterile culture from explant tissue, 2) multiplication of shoots from the explant, 3) development of roots on shoots to produce plantlets, and 4) production of self-sufficient plants through hardening off. The objectives are to set up a micropropagation system through each stage using African violet. Various procedures are described for each stage, including media preparation and observations recorded. Results showed prolific bud induction and higher multiplication rates using smaller sub-cultured tissue pieces. Root induction was near 100% with one medium.
The general procedure for plant tissue culture involves sterilizing glassware and tools, preparing and sterilizing explant tissue samples, producing callus growth from the explants on nutrient media, proliferating the callus through subculture, and establishing suspension cultures. Key steps include surface sterilizing explants using chemicals like sodium hypochlorite, transferring sterilized explants to growth media, incubating to produce initial callus, subculturing callus periodically to fresh media, and creating suspension cultures by transferring callus to liquid shaking media.
The document discusses plant protoplast isolation, purification, and culturing. Some key points:
- Protoplasts are plant cells that have had their cell walls removed, leaving just the plasma membrane. They allow for plant cell fusion and regeneration.
- Protoplasts are typically isolated from plant tissues like leaves using enzymatic digestion with cellulase and pectinase. This yields more protoplasts than mechanical methods.
- Isolated protoplasts are purified by centrifugation and washing to remove cell debris. They are then cultured in liquid or solid nutrient media and tested for viability before regeneration.
Protoplasm fusion involves removing the cell wall from plant or bacterial cells through enzymatic digestion to create protoplasts. Protoplasts can be fused through spontaneous fusion during isolation or induced fusion using various methods like mechanical manipulation, chemical fusogens, or electricity. Successful fusion results in a hybrid cell containing genetic material from both parent cells. Protoplast fusion allows for combining genes from sexually incompatible species and is useful for plant breeding and genetic engineering applications.
Transformation of tomato using CRISPER- CAS9 construct to enhance the shelf l...ShreyasGowda87
This document summarizes a student project to transform tomato plants using CRISPR-Cas9 to enhance shelf life. The objectives are to standardize tomato hypocotyl regeneration and optimize genetic transformation using a CRISPR construct targeting two tomato pectate lyase genes. The methodology involves germinating tomato seeds, taking hypocotyl explants, co-cultivating with Agrobacterium containing the CRISPR construct, regenerating plants on selection media, and rooting regenerants to obtain transgenic plants with increased shelf life. Preliminary results showed regeneration of putative transgenic shoots from several batches of explants.
This document outlines the steps for conducting an ethnobotanical study and screening collected plants. It involves selecting an unexplored study area, collecting ethnobotanical knowledge from local people, classifying and preserving collected plant species, screening 10 plants for further analysis using qualitative and quantitative methods, and extracting, amplifying, and detecting DNA from samples. The goal is to systematically evaluate traditional uses of plants and characterize the biochemical and molecular basis of these uses.
This document discusses cytogenetics and chromosome analysis techniques. It begins with an introduction to human chromosomes and chromosomal abnormalities. It then describes various types of chromosomal mutations and abnormalities that can be detected through karyotyping and fluorescence in situ hybridization (FISH). The document provides detailed procedures for chromosome sample preparation from bone marrow and blood cultures, as well as staining and analysis techniques like Giemsa staining and G-banding. The importance of chromosomal studies for diagnosing conditions like Turner syndrome and Klinefelter syndrome is also highlighted.
This document describes procedures for plant regeneration through callus culture using tobacco plants. It involves initiating callus cultures from tobacco explant tissue, observing the growth pattern of callus cultures over multiple phases, and inducing shoot and root organogenesis from the callus to regenerate tobacco plants. The objectives are to initiate and maintain callus cultures from tobacco explants, create cell suspension cultures, and induce organ development from callus to regenerate whole plants. Standard procedures are provided for callus initiation, maintenance, measurement of growth curves, and regeneration of shoots and roots. Results showed high rates of callus formation and organogenesis from explants and callus on optimal media formulations.
This document provides a detailed tutorial on the procedure for culturing human melanocytes. Key steps include:
1) Isolating epidermis from skin specimens using dispase enzyme solution overnight at 4°C.
2) Dispersing epidermal cells using trypsin and mechanically dissociating into a single cell suspension.
3) Seeding cells in TPA-free growth medium and incubating without disturbance for 48-72 hours.
4) Maintaining cultures by changing medium twice weekly and passaging confluent cultures using trypsin.
Methods for cryopreserving and thawing melanocytes are also described. Morphology and growth characteristics of cultured melanocytes are provided.
This document provides instructions for performing an enzyme-linked immunosorbent assay (ELISA) to quantitatively detect total aflatoxins (B1, B2, G1, and G2) in grains, nuts, and other commodities. Aflatoxins are toxic metabolites produced by certain molds that can contaminate foods and animal feeds. The ELISA uses an aflatoxin-specific antibody to competitively bind sample aflatoxins or HRP-conjugated aflatoxin. The intensity of the color reaction indicates the aflatoxin concentration in samples, which can be quantified by comparison to standard curves. Validation studies showed the assay can reliably detect aflatoxins from 1-20 p
This document provides instructions for using an ochratoxin A assay kit. It describes ochratoxin A as a mycotoxin produced by molds that is toxic and carcinogenic in humans and animals. The kit is intended to quantitatively detect ochratoxin A in grains, coffee, cocoa, spices and other foods. It works by competitively binding ochratoxin A from standards and samples to an antibody, and then detecting the amount of bound conjugate using a colorimetric reaction. Detailed procedures are provided for extracting ochratoxin A from different sample types and running the assay.
This document discusses the process of tissue processing in histopathology. It describes the key steps which include:
1. Tissue collection, fixation to prevent decay, and gross examination.
2. Processing which involves dehydration, clearing, infiltration with paraffin wax, and embedding tissues in paraffin blocks for sectioning.
3. Sectioning tissues with a microtome and staining, typically with hematoxylin and eosin, for microscopic examination.
This presentation gives all the basic necessary details about cell culture and karyotyping in cell lines . It describes the important methods like subculture,confluency,cell count,passaging and karyotyping in cell culture.It also describes about BSL.It also gives a brief idea about types of cell culture.This presentation also includes the requirements in the cell culture lab
This presentation deals tissue processing in histopathology, the detailed of presentation given blow:
Histology, study the organization of tissues at all levels, from the whole organ down to the molecular components of cells that are found in most multicellular plants and animals.
Animal tissues are classified as epithelium, with closely spaced cells and very little intercellular space; connective tissue, with large amounts of intercellular material; muscle, specialized for contraction; and nerve, specialized for conduction of electrical impulses. Blood is also sometimes considered a separate tissue type.
Plants are composed of relatively undifferentiated tissue known as meristematic tissue; storage tissue or parenchyma; vascular tissue; photosynthetic tissue or chlorenchyma and support tissue or sclerenchyma and collenchyma.
nettle propsal presentation power point Afaf final copy.pptxAfafAbuhilal
This document presents a literature review and methodology for a study on the extraction of bioactive compounds from stinging nettle (Urtica dioica). Key points:
1. Stinging nettle is a common weed with stinging hairs that contains various nutrients, minerals, vitamins, proteins, and polyphenols like ursolic acid and quercetin that have medical benefits.
2. The study will extract bioactive compounds using solvent extraction methods like Soxhlet extraction and determine total phenolic content and antioxidant activity. It will also examine antimicrobial effects.
3. A literature review found that ethanol extraction via Soxhlet had the highest yields in other studies. Parameters like solvent, temperature
This document describes the microbial limit test, which includes tests to quantify and qualify microorganisms in samples. It involves estimating total viable counts of bacteria and fungi, and detecting specific pathogens. The test is based on culturing samples on various media to support or inhibit growth of target microbes. Methods like membrane filtration, spread plating, and serial dilution are used to quantify microbes, while selective media help identify pathogens like E. coli, S. aureus, P. aeruginosa, and Salmonella. Detailed procedures are provided for quantification, enrichment, and identification of microorganisms in samples.
Human liver microsomes & rat liver microsomesgaurav sharma
Human and rat liver microsomes are subcellular fractions containing cytochrome P450 enzymes and other drug-metabolizing enzymes. They are commonly used in vitro to study drug metabolism and interactions. Human liver microsomes are obtained from human liver tissue through differential centrifugation and contain enzymes for phase I and phase II drug metabolism. They are useful for identifying drug metabolites, evaluating interspecies differences, predicting in vivo clearance, and studying interindividual variability in drug metabolism. Rat liver microsomes were also discussed as an experimental model. Incubation methods and analytical techniques like HPLC were described for evaluating metabolism using liver microsomes.
The extraction of DNA involves three main steps that are cell lysis, protein separation, and DNA purification. Cell lysis is usually performed by incubation of cell in buffer containing detergent and protease. Cellular proteins are salted out or phase separated using organic solvents. Finally DNA is isolated and purified either by alcohol precipitation or adsorption with silica and elution.
1) The document summarizes different types of cell viability assays, including dye exclusion assays, colorimetric assays, fluorometric assays, and luminometric assays.
2) Specific assays discussed in more detail include trypan blue exclusion assay, MTT assay, SRB assay, and 5-CFDA-AM fluorometric assay.
3) The assays are described in terms of their principles, protocols, calculation methods, and applications in measuring cell viability and screening drug responses.
Nguyen Thi Nhi completed the following works in the third week of her master's program:
1) Learned techniques for MIN6 cell culture including making media and using an autoclave.
2) Studied cell culture and immunoassay techniques in theory.
3) Specifically learned how to make cell culture media including DMEM, FBS, penicillin/streptomycin, and beta-mercaptoethanol and the purpose of each component. She also learned proper operation of the JSR autoclave for sterilization.
Similar to Protoplast Isolation and Culture.pptx (20)
This document discusses bead-based separation techniques for isolating biomolecules like DNA. It explains that magnetic beads containing iron oxide particles can selectively bind to target molecules like DNA under certain buffer and salt conditions. The DNA is then separated and purified from other contaminants by applying a magnetic field to pull the beads and attached DNA out of solution while leaving behind other molecules. The DNA is later released from the beads in low salt buffer for downstream applications. The technique allows for rapid separation and purification of nucleic acids without centrifugation or filtration.
The document discusses the process of micropropagation through adventitious shoot proliferation using African violet as an example. There are four main stages: 1) initiation of sterile culture from explant tissue, 2) multiplication of shoots from the explant, 3) development of roots on shoots to produce plantlets, and 4) production of self-sufficient plants through hardening off. The objectives are to set up a micropropagation system through each stage using African violet. Various procedures are described for each stage, including media preparation and observations recorded. Results showed prolific bud induction and higher multiplication rates using smaller sub-cultured tissue pieces. Root induction was near 100% with one medium.
Gene cloning involves isolating a gene from an organism's DNA, inserting it into a vector, and transforming host cells to generate multiple copies of the gene. Several techniques can confirm successful cloning, including DNA sequencing to verify the nucleotide sequence, restriction enzyme analysis to check for specific DNA fragments, and functional assays to test protein activity if the gene encodes a protein.
Transformation is a process of horizontal gene transfer where foreign DNA is inserted into bacterial cells. It involves making bacterial cells competent, isolating foreign DNA, mixing cells with DNA, incubation, expression of new traits, and selection. Transformation is widely used in research to produce proteins and study bacterial evolution.
Plant transformation introduces specific genes into plants using various techniques like Agrobacterium-mediated transformation or particle bombardment. The process involves selecting a plant species and gene of interest, constructing a vector, introducing the new gene into plant cells, selecting and regenerating transformed cells, validating gene presence and expression, and field testing if needed. It is an important biotechnology method but requires following ethical and regulatory standards.
ELISA is an immunological technique that detects the presence of an antigen or antibody using an enzyme-linked reaction. It has high sensitivity and specificity. There are four main types of ELISA - direct, indirect, competitive, and sandwich - which differ in their use of primary and secondary antibodies and whether the antigen or antibody is immobilized. ELISA is a widely used, inexpensive, and relatively quick technique for detecting various infections and antigens, with advantages over radioimmunoassay including lack of radiation hazards.
The document discusses SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), a technique used to separate proteins based on their molecular weight. SDS denatures proteins and gives them a uniform negative charge, allowing separation based primarily on size in the polyacrylamide gel. The document provides details on preparing the resolving and stacking gels, running the electrophoresis, and using SDS-PAGE to separate and analyze proteins.
This document describes how to quantify DNA and RNA concentrations using a UV-visible spectrophotometer. It explains that spectrophotometry uses Beer's Law and Lambert's Law to relate absorbance to concentration and path length. The procedure involves preparing samples, taking blank and sample measurements at 260nm, and using the measured absorbances and dilution factors to calculate concentrations according to the Beer-Lambert Law. Purity is also assessed by calculating absorbance ratios.
Agarose gel electrophoresis is a technique used to separate macromolecules like DNA, RNA, and proteins based on their size. It involves dissolving agarose powder in buffer solution to form a gel matrix in a mold. Samples are loaded into wells in the gel and an electric current is applied, causing the charged molecules to migrate through the pores at different rates according to their size. After electrophoresis, the gel is stained and visualized under UV light to see separated bands of the macromolecules. This technique is widely used in molecular biology for applications such as DNA fragment sizing and PCR confirmation.
The document discusses techniques for isolating genomic DNA from different biological samples. It describes the general procedure as lysing cells to release DNA, precipitating proteins and cell membranes, and isolating the DNA using alcohol precipitation. Specific procedures are provided for isolating DNA from blood, tissues, cultured cells, bacteria, and plants. The procedures involve lysing the source material through various mechanical or chemical means, then purifying and collecting the DNA through precipitation and resuspension in buffer solutions.
Ion exchange chromatography is a technique used to purify charged biomolecules like proteins, peptides, and nucleic acids. It works by exploiting the electrostatic interactions between ionized functional groups on a stationary phase and the molecules in the sample. There are two main types: cation exchange uses negatively charged groups like sulfonic acid to attract positively charged cations, while anion exchange uses positively charged groups like amino to attract negatively charged anions. Samples are loaded onto the column in a compatible buffer and then different components are selectively eluted by adjusting the pH or ionic strength to disrupt the electrostatic bonds between molecules and stationary phase.
This document discusses paper chromatography and thin layer chromatography. Paper chromatography uses cellulose filter paper as the stationary phase and a liquid as the mobile phase to separate solid and liquid compounds based on their polarity. Thin layer chromatography uses a silica gel coated glass plate as the stationary phase and a liquid mobile phase to separate non-volatile mixtures based on the polarity of particles toward the phases. Both techniques work on the principle of separating compounds based on their distribution between a solid stationary phase and liquid mobile phase.
Fractionation and centrifugation are techniques used to isolate and separate biological components from tissues. The process involves homogenizing tissues to break cells and release contents. The homogenate is subjected to differential and density gradient centrifugation, which separate components based on density and size. This allows organelles and other structures to be isolated in different fractions for further analysis and study of their structure and function.
This document outlines the key concepts and techniques covered in a biotechnology course, including 12 lectures. The first lecture defines buffers and the Henderson-Hasselbalch equation for calculating pH. It describes how to prepare buffer solutions using a weak acid and its conjugate base. The lecture also explains pH measurement using a pH meter, pH paper, or universal indicator and the importance of calibration and electrode care.
Y-STR haplotype databases are essential for forensic DNA analysis and allow researchers to study paternal lineages. They contain genetic profiles based on variations in short tandem repeats on the Y chromosome. These databases can be used for paternity testing, forensic identification by matching crime scene DNA to suspects, identifying remains in missing persons cases, and population genetics research. Maintaining high quality standards is important for the reliability of matches between evidence and database profiles.
This document discusses lineage markers that can be used to trace maternal and paternal lineages. It explains that mitochondrial DNA is passed down unchanged from mothers to children and can be used to determine maternal lineage. The Y chromosome is passed from fathers to sons and its DNA markers can trace paternal lineage. While lineage markers only provide information about one ancestral line, they are still useful in forensic investigations when autosomal DNA is limited or degraded.
Y chromosome DNA testing can be used to investigate male lineage and paternal origins. There are three main types of Y chromosome DNA testing: 1) Y-STR testing examines short tandem repeats to generate a unique genetic fingerprint for identifying recent male ancestry or paternity. 2) Y-SNP testing examines single nucleotide variations to reveal details about historical migration patterns and haplogroup. 3) Big Y testing sequences a large number of markers including SNPs and STRs to provide a thorough understanding of a person's Y chromosome and further trace their lineage back in time. Y chromosome testing is often used along with autosomal DNA testing to create a more complete picture of a person's genetic history.
Forensic DNA samples often contain degraded DNA and inhibitors that make analysis difficult. Environmental exposure degrades DNA, while substances like hemoglobin or dyes can inhibit PCR. To address this, samples may be diluted to reduce inhibitors, or more polymerase can be added to overcome them. Mixed samples and low copy number DNA present additional challenges, like detecting minor components or dealing with stochastic effects. Proper controls and replication are needed to reliably interpret low level DNA results.
This document discusses 7 common methods used in forensic DNA analysis: 1) PCR is used to amplify DNA segments of interest like STRs. 2) STR analysis determines the lengths of short tandem repeats to identify individuals. 3) SNP analysis identifies single nucleotide polymorphisms for population studies and identification. 4) mtDNA sequencing analyzes maternal ancestry when nuclear DNA is degraded. 5) DNA methylation analysis assesses epigenetic patterns for tissue identification and age determination. 6) NGS allows high-throughput sequencing for genomic analysis. 7) Capillary electrophoresis separates and examines DNA fragments by size. These techniques are often combined to produce a precise DNA profile for identification.
Population variation is an important consideration in forensic science and DNA evidence interpretation. Understanding genetic diversity across populations allows for more accurate assessment of DNA profile matches and inferences about sample origins. Factors like ancestral genetic markers, allele frequencies in databases, subpopulation structures, and statistical analysis techniques all require awareness of population genetics to avoid imprecise or prejudiced conclusions from DNA evidence. Emerging technologies continue to provide more detailed analysis of population variation.
Polymerase chain reaction (PCR) is used in several forensic DNA typing methods to amplify specific regions of DNA. These include STR analysis of microsatellite repeats, SNP analysis of single nucleotide variations, mtDNA analysis for maternal lineage, and Y-chromosome analysis for paternal lineage. PCR amplifies targeted regions which are then analyzed using techniques like capillary electrophoresis to develop a DNA profile that can identify individuals or determine ancestry.
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Denis is a dynamic and results-driven Chief Information Officer (CIO) with a distinguished career spanning information systems analysis and technical project management. With a proven track record of spearheading the design and delivery of cutting-edge Information Management solutions, he has consistently elevated business operations, streamlined reporting functions, and maximized process efficiency.
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Date: May 29, 2024
Tags: Information Security, ISO/IEC 27001, ISO/IEC 42001, Artificial Intelligence, GDPR
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Training: ISO/IEC 27001 Information Security Management System - EN | PECB
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2. Introduction
• Protoplasts can be isolated from plant tissues or cultured cells
by enzymatic digestion to remove the cell walls.
• The enzymes for this purpose are preparations which are
commercially available.
• The success of protoplast isolation depends especially on the
condition of the tissue and the combination of enzymes being
used.
• Methods and procedures for protoplast isolation from plant
tissues have long been known.
• Recent advances in the isolation, culture, and regeneration of
plants from protoplasts of a wide diversity of species have
been reported.
3. Objectives and Goals :
• To present the technique for isolation of protoplasts at high
yield from stem cortex tissues of Brassica napus (canola) and
an intergeneric hybrid of tomato.
• To present general procedures for the isolation of protoplasts
from cell suspension cultures.
• To provide procedures for protoplast culture, and to observe
cell wall reformation and cell division in such cultures.
4. Equipment and Supplies :
• Autoclave or steam sterilizer (pressure cooker)
• Laminar air flow hood, Canadian Cabinets or equivalent
• Clinical centrifuge, IEC Centra 4B or equivalent, Fisher Scientific
• Gyratory or orbital shaker
• Conical or round bottom tubes, 50 ml glass or plastic
• Inverted microscope, Zeiss 1M or equivalent
• Filtration filter units, Millipore 0.2 f.Lm membranes
• Incubator or growth room, with temperature, light, and humidity controls
• Fine forceps and sterile scalpels
• Erlenmeyer flasks, 125 ml
• Funnels, 55 mm diameter
• Sterile wide-mouth pipettes, glass or plastic
• Pasteur pipettes with medium bore size (Note. Cut standard Pasteur
pipettes to obtain a bore diameter of about 3 mm) .
5. jh
• Hemacytometer
• Sterile petri dishes, 100 X 15mm, 60 X 15mm
• Nylon mesh filters, 64 and 44 f.Lm pore sizes
• Calcium hypochlorite
• Agarose, SeaKem LE or equivalent purity, FMC BioProducts or Sigma Co -
Gelrite or Phytagel, Sigma Co - Macerozyme R-10, Yakult Honsha Co Ltd,
Tokyo, Japan
• Cellulase R-lO, Yakult Honsha Co Ltd, Tokyo, Japan
• Pectolyase, Sigma Co (effective pectinase, not used in the protocols
6. Procedures:
• Enzyme Stock Solutions : Each is prepared by
dissolving in 0.4 M mannitol (72.8 gil) at pH 5.8,
stored frozen at -20 DC:
• Macerozyme R-10, 10% w/v, O.lg in 10ml.
• Cellulase R-10, 10% w/v, 0.1 gin 10ml.
• When needed, the enzyme stocks are diluted to the
desired concentration in 0.4 M mannitol and
sterilized using 0.22 !-tm filters.
• Donor Plant Materials : Procedures are described for
the isolation and culture of stem cortical tissue
protoplasts of Brassica species (Klimaszewska and
Keller 1987) and a Lycopersicon intergeneric hybrid.
7. h
• Growth of Donor Plants of Brassica napus (Canola, Oilseed
Rape) : Seeds of cv. Westar are obtained from the Agriculture
Canada Research Station, Saskatoon, SK, Canada.
• Protoplasts are produced in good yields from stem cortex
tissues of this genotype. The protoplast isolation, culture, and
plant regeneration procedures can possibly be adapted to
other cultivars and varieties of B. napus.
• Plants are grown in 150-cm diameter fiber pots in a
soil:sand:peat (2: 1: 1) mixture under a 16/8 h day/night
photoperiod, 360 ftmol m- 2 S-I, at 20/ 15°C. Plants are
watered daily and receive weekly fertilizer with a 20:20:20 N:
P: K liquid nutrient solution at 1 gIl.
• Good quality, vigorous, healthy plants are essential for the
success of protoplast culture to achieve sustained cell division
and plant regeneration.
8. Preparation of Explants :
• Excise three to four terminal internodes from B. napus cv. Westar
plants at the early flowering stage (three to five flower buds open
per plant), or from the intergeneric hybrid of tomato.
• Surface sterilize in 7% w/v of saturated calcium hypochlorite
solution for 10 min and rinse the internodes in sterile distilled water
3 X for 5 min each.
• A sterile plastic petri dish, 100 X 15 mm, is opened and used as a
sterile tray to cut the tissues from the stem sections.
• Peel off epidermal and subepidermal cell layers from the stems
using sterile thin scalpel blades and fine jeweller's forceps. A good
explant is 3-6 mm long, depending upon the plant species.
• The epidermal segments are precultured in the dark at 4°C
overnight in lOIS ml of liquid MS medium in 100 X 15mm petri
dishes.
9. Protocols :
• Enzymatic Digestion to Release Protoplasts :
• The pretreated epidermal segments are transferred
to 100 X 15-mm petri dishes containing 10-15 ml of
filter sterilized enzyme solution consisting of 1%
(w/v) Macerozyme R-lO and 1% Cellulase R-lO in 0.4
M mannitol, pH 5.8.
• Seal the petri dishes with parafilm and incubate for
16 h at 28°C in the dark on a slow gyratory shaker.
10. Protoplast Filtration and Washing (Purification) :
• . Gently transfer the protoplasts, enzyme solution, and
undigested tissues with a sterile, medium-bore Pasteur
pipette onto a sterile 64 or 44 ~m pore-size filter inside a
sterile funnel.
• Collect the filtrate in a sterile 50 ml centrifuge tube, cover
with sterile tin foil or autoclaved metal closure, and centrifuge
at 500 g for 5-10 min.
• Remove the enzyme solution carefully with a sterile Pasteur
pipette.
• Add 12 ml of 0.4 M sucrose to the pellet, and gently disperse
and resuspend the protoplasts in the solution. Use gentle with
drawing and delivery with the pipette.
11. r
• When protoplasts are completely dispersed, add carefully
with a Pasteur pipette, by layering over the sucrose solution: 3
ml of a solution of 0.4 M sorbitol, 10 mM CaCI2 • 2HP, 5 mM
H MES, pH 5.8, and centrifuge at 250 g for 6min.
• To wash the protoplasts, use a Pasteur pipette to collect the
protoplasts carefully at the interphase of the two solutions,
and transfer to 4 ml of salt solution containing 0.2% w/v CaCl2
and 2.5% KCI, pH 6.0. Gently suspend and centrifuge at low
speed to collect the protoplasts. Remove the washing solution
carefully with a sterile Pasteur pipette.
12. j
• Resuspend the protoplasts in the centrifuge tubes to a density
of 1 X 105 per ml in L Medium (Klimaszewska and Keller 1987)
for Brassica and in SCM1 Medium (Tan et al. 1987) for tomato.
To determine the density, a hemacytometer and inverted
microscope are used to make a count of the protoplasts.
• Count the protoplasts in the hemacytometer. Count 64 fields,
each being 0.25mm X 0.25 mm (see Fig. 1). Calculate the
average number of protoplasts per field and per ml. Because
each field is 0.25 mm X 0.25 mm X 0.1 mm (depth), or 6.25 X
10-3 mm each field is 6.25 X 10-6 ml.
13. Protoplast Culture :
• The protoplasts may be cultured in one of three ways:
• as thin liquid layers of 1-1.5 ml per 60 X 15 mm sterile
petri dish;
• as micro droplets of 10-25 III each, spotted in the petri
dishes
• as thin liquid layers of 1.5 ml over agarose underlayers.
• Agarose underlayers consist of 2 ml of the respective culture
medium solidified with 0.4% (w/v) agarose, in the bottom of
60 X 15 mm petri dishes. L Medium is the resuspension and
culture medium for Brassica protoplasts while the SCM-l
Medium is the resuspension and culture medium for hybrid
tomato protoplasts.