...LAB#11 DNA EXTRACTION & KARYOTYPING ________________________________________________ Objectives: After completing this exercise, you should be able to: 1. Develop an understanding of the structure and properties of DNA, based on observation and manipulations 2. Understand some implications of DNA technology 3. Extract DNA from an onion to understand that all cells contain DNA Introduction/Purpose: DNA is too small to see under a regular microscope, so then how can it be studied? DNA is a large chemical molecule found in all living things, so it should be possible to extract it from cells or tissue. All we need to do is disrupt the cell’s plasma membrane and nuclear envelope, make the DNA clump together. DNA extraction is possible. Plant material is easy to use and DNA extractions from onion, bananas, liver, or wheat germ are common classroom activities or demonstrations. Plants used in agriculture and horticulture are often artificially selected for their large flowers and fruits. Strawberries are no exception. A reason for the size of today’s large supermarket strawberries is the octaploid nature of their cells. With eight sets of chromosomes, they have plenty of DNA for classroom extraction. The fruit is homogenized with a detergent to prepare a filtrate. The detergent emulsifies and forms complexes with the lipids and proteins of the plasma membrane this causes them to precipitate out of the solution. The mixture is then filtered through cheesecloth;...
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...|Do-It-Yourself Strawberry DNA LAB | |Introduction: [pic] | |Since DNA is the blueprint for life, everything living contains DNA. DNA isolation is one of the most basic and essential | |techniques in the study of DNA. The extraction of DNA from cells and its purification are of primary importance to the field of | |biotechnology and forensics. Extraction and purification of DNA are the first steps in the analysis and manipulation of DNA that | |allow scientists to detect genetic disorders, produce DNA fingerprints of individuals, and even create genetically engineered | |organisms that can produce beneficial products such as insulin, antibiotics, and hormones. | |DNA can be extracted from many types of cells. The first step is to lyse or break open the cell. This can be done by grinding a | |piece of tissue in a blender. After the cells have broken open, a salt solution such as NaCl and a detergent solution containing | |the compound SDS (sodiumdodecyl sulfate) is added. These solutions break down and emulsify the fat & proteins that make up a cell| |membrane. Finally, ethanol is added because DNA is soluble in water. The alcohol causes DNA to precipitate, or settle out of the | |solution, leaving behind all the cellular...
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...Pepper Seed DNA Extraction Biochem lab: CHE 452L marisol gomez Pepper Seed DNA Extraction Biochem lab: CHE 452L marisol gomez 2015 2015 INTRODUCTION The jalapeno is a member of the capsicum family, along with many other peppers. The usual methods for characterization of different pepper species are based on their morphological and physiological traits, however this many not always be enough. For peppers, their traits are influenced by things like their genotype or their specific environment. Genomic markers can allow for a more direct comparison of closely related individuals (Ansari and Khan, 2012). In our case we focus on DNA extraction. The two basic parts of a DNA extraction procedure include the breaking of the cell walls to expose the DNA and the use of enzymes to remove contaminants. The DNA is analyzed for purity by taking the absorbance. The pure DNA is then visualized by gel electrophoresis. The DNA extraction of plant seeds is difficult because of their cell wall. The method used to break the cell wall includes grinding the seeds with liquid nitrogen. The addition of DNAzol is used to isolate genomic DNA (Chomczynski et al. 1997). Restriction enzymes are necessary to fragment patterns of the DNA and in turn making it easier to analyze the DNA through gel electrophoresis. BACKGROUND The purpose of our experiment is to extract the DNA from pepper seeds to be able to compare and contrast the similarities in their DNA. The extraction of DNA from a plant...
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... Title: Extraction of genomic DNA Introduction: Isolation of genomic DNA is one of the most commonly used practices in several areas of science. Purified, high molecular weight DNA is used for many applications such as: * To prepare genomic DNA libraries * To isolate genes to be cloned * To carry out southern blotting and hybridization Many different methods are available for isolation of genomic DNA, and they all involve the following basic stages. * Disruption and lysis of starting material: * Cell walls and cell membranes are broken to release DNA and other cellular components. * Removal of proteins and other contaminants: * Protein contaminations are removed by digestion with proteinase K followed by salting out and denaturing some proteins by SDS. * Recovery of DNA: * DNA is precipitated using ethanol or isopropanol in the presence of monovalent cations All DNA isolation protocols have a common goal in isolating high molecular weight DNA, which meet the following three criteria: 1) Purity should be high enough for further downstream applications such as restriction digestion. 2) DNA should be intact to give accurate and reproducible migration patterns on gel electrophoresis. 3) Yield should be high enough to obtain a required quantity of DNA from a reasonable amount of tissue. Materials and Methods: Materials Bench centrifuge...
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...Extracting DNA from a Strawberry Preparation/Set-Up Place ethanol in freezer prior to experiment and allow to stand until cold. Prepare a container for the DNA by wrapping a small tray with saran wrap. Preparing an Extraction Buffer Fill a container with approximately 900 mL of water Add 50 mL of dish washing soap or shampoo (DO NOT use any shampoo containing conditioner) Add 2 teaspoons of salt Slowly Invert container and allow contents to swirl - This section will contain a graphic of the type of tube that will hold the buffer Preparing your Filtration Apparatus Obtain a funnel and cheesecloth Drape the cheesecloth over the edge of the funnel, securing firmly Place the tube of the funnel into a 50 mL glass vial - A graphic will be inserted showing the completed apparatus Procedure Preparing the Strawberries Obtain 2 to 3 strawberries and remove all stems and leaves Place strawberries in a zip lock bag Seal ziplock bag tightly, allowing no air to remain Use your fingers to squish the strawberries. Continue with this step for two minutes or until strawberries are completely reduced to paste. -Graphic here to illustrate process of squeezing bag Adding the Buffer Add approximately 3 tablespoons of the buffer to the zip lock bag and then reseal tightly, being sure to allow no air to remain Use your fingers to squish the mixture, allowing the buffer to distribute evenly for 1 minute ...
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...Tooth loss and dental extractions have existed for centuries with the latter being the main tool for providing relief from dental pain and removing diseased tissues (Torabinejad). There was an estimated 50 million dental extractions performed in the United States in 1979 (Bullock). With the increased number of people living in the United States for a longer period of time, the occurrence of dental extractions and tooth loss has certainly risen exponentially. Unfortunately, tooth loss, whether it is through dental extractions or other means, has long term clinical sequelae associated with it. Osteonecrosis of the jaw, bacteremia, orbital cellulitis, and other psychological and physiological clinical sequelae that impact the well-being of the patient will be discussed. Osteonecrosis of the jaw (ONJ) is defined as the presence of exposed bone in the mouth that fails to heal after appropriate intervention over a period of 6 to 8 weeks (Reid), and results in chronic osteomyelitis with areas of bone necrosis. Most commonly affecting the mandible (Bagan), patients with ONJ experience symptoms that range from painless exposed bone to severe jaw pain (Sambrook). The majority of cases of ONJ have been found to be initiated and associated with tooth extraction procedures as a result of the introduction of oral flora to the exposed jaw bone that prevents healing and becomes infected (Ruggiero). In addition to tooth extractions being a precipitating event to ONJ, it has been found...
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...population of an elliptical at the Dixon Recreation Center by analysis of DNA from an uncultured community sample. 16S rRNA genes will distinguish bacteria and represent the microbial diversity of the sampled elliptical. An estimate of the cleanliness of the elliptical will be made based on species-abundance as well as evidence of any pathogenic strains. We will also compare and contrast respective populations of bacteria on clean and disinfected ellipticals to assess disinfectant effectiveness. Methods We collected bacteria from the elliptical, purified and amplified the total community DNA, and then analyzed the sequenced DNA with Mi Seq Software. A revised protocol was used to extract, purify, and sequence the community DNA; these methods were followed without deviation (1). The procedure to sequence and configure the data is described on Blackboard (2). Results PCR amplification of the community DNA product was unsuccessful. There was no evidence of PCR amplified DNA on the gel (Figure 1.1). An alternate gel from Molecular Microbiology Laboratory was used as a model to perform gel electrophoresis of the community DNA (2). A plot of migration distance vs. size that includes the Invitrogen ladder can be used to determine the size of the PCR amplified community DNA (Figure 1.2). [Figure 1.2] Base Pairs vs. Migration Distance of Invitrogen Low DNA Mass Ladder. The distance these standardized fragments is associated...
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...DNA Technology BIO/240 May 6, 2013 DNA Technology INTRO – LEE DNA Technology: Cloning, Gene Therapy, and Stem Cell Research DNA technology encompasses a wide variety of applications and because of the duplicating nature of DNA, it is easy to see how humans could benefit from its manipulation. One such technology is cloning. Cloning technology comes in three forms: recombinant DNA cloning, reproductive cloning, and therapeutic cloning. Cloning Recombinant DNA cloning consists of transferring DNA fragments from an organism to a self-replicating element, like a bacterial plasmid. The fragments join with the cloning vector and are reproduced with the host cell. This technology is most commonly known for its use in genetically modified foods. DNA fragments that code for better tasting, higher nutrient qualities are spliced into regular plants to produce super foods (US Dept of Energy Genome Program, 2009). Reproductive cloning takes all the genetic information out of a cell and replaces it with DNA from the desired organism. With luck, this cell will begin to divide until it becomes an embryo and can be implanted into a host mother (US Dept of Energy Genome Program, 2009). Gene Therapy and Stem Cell Research Therapeutic cloning is by far the most controversial. This type of cloning produces human embryos for use in research, and usually for the stem cells that can be harvested from these embryos. Stem cells can be used to clone organs and body parts from the...
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...Laboratory Exercise #8 DNA Fingerprinting: Identification of DNA Restriction Fragmentation Patterns I. Introduction All humans have in common the coding sequences of their DNA, but, unless you are an identical twin, the non-coding sequences of your DNA are like no other person’s on the planet. The bulk of human DNA does not code for specific genes and is highly repetitive. A British geneticist, Alec Jefferies, developed laboratory techniques in 1984 that became known as DNA fingerprinting. These techniques can identify the differences in repetitive nucleotide sequences between individuals, but also show where sequences are the same and, therefore, have been inherited. DNA fingerprinting can be used to detect genetic disorders, identify individuals, settle paternity disputes and determine guilt or innocence when presented as evidence in a crime scene investigation. This ability to identify an individual is enhanced by the variety of substances that contain DNA, including blood, semen, saliva, hair, urine, bone, teeth, feces, and tissues. DNA profiling is one of the most important applications of the techniques used in the field of forensics. Crime scene investigation routinely includes the collection of evidence that may contain DNA in the hopes that it may provide a link to a suspect. The genetic comparison doesn't require that investigators look at the entire genome of the DNA samples found at the crime scene or from a suspect. Instead, forensic scientists use genetic...
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...PATHOGENESIS 2 1.1.4 TREATMENT 3 1.2 MOLECULAR BIOLOGY 3 1.2.1 STRUCTURE OF GENOME 3 1.2.2 GENETIC VARIATION 6 1.2.3 GENOTYPIC DIFFERENCES 8 1.3 RNA DEPENDENT RNA POLYMERASE ACTIVITY 9 1.3.1 POLYMERASE FUNCTION 9 1.3.2 MODEL SYSTEMS OF HCV REPLICATION 11 1.3.3 GENOTYPE SPECIFIC STUDIES 11 1.3.4 BIOCHEMICAL PROPERTIES 12 1.4 KUNJIN VIRUS RNA DEPENDENT RNA POLYMERASE 13 1.5 CONCLUSION 15 1.6 AIMS AND HYPOTHESIS 16 2 MATERIALS AND METHODS 17 2.1 HCV POSITIVE SERA SAMPLES 17 2.2 RNA EXTRACTION 17 2.3 CDNA SYNTHESIS 17 2.4 HCV PRIMER DESIGN AND USAGE 18 2.5 NESTED POLYMERASE CHAIN REACTION (NPCR) 21 2.5.1 REACTION AND CYCLING CONDITIONS 21 2.5.2 PCR PRODUCT PURIFICATION 22 2.6 AGAROSE GEL VISUALISATION 22 2.7 DNA SEQUENCING 22 2.8 DNA SEQUENCE AND PHYLOGENETIC ANALYSIS 23 2.9 KUNJIN VIRUS PLASMID 23 2.10 KUN PRIMER DESIGN AND USAGE 23 2.11 CLONING PCR PRODUCTS 24 2.11.1 RESTRICTION DIGEST 24 2.11.2 LIGATION 24 2.11.3 TRANSFORMATION 24 2.11.4 COLONY PCR 24 2.12...
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...the discretion of the Assessment Board. | Internal verification: | Date: | | Name | | | | Signature | | Aim and purpose:-To develop understanding of the principles of Mendelian genetics and to develop knowledge and practical techniques used in commercial, analytical and research laboratories | | GRADING CRITERIA To achieve a pass grade the evidence must show that the learner is able to: | To achieve a Merit grade the evidence must show that the learner is able to: | To achieve a distinction grade the evidence must show that the learner is able to: | P1 Compare and contrast the structure of various nucleic acids. | M1 Explain how genetic information can be stored in a sequence of nitrogenous bases in DNA. | D1 Explain the steps involved in biosynthesis of protein including the roles of...
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...purification of genomic DNA requires three main processes which include, lysing of cells to release the DNA, purifying the solution by removing unwanted macromolecules, and precipitating the DNA from the solution. The process of isolation and purification requires several reagents. The Nuclei Lysis Solution contains lysozyme and EDTA. The lysozyme is an enzyme that degrades peptidoglycan, which is a rigid exoskeleton cell wall that protect the bacteria (3). Disrupting the peptidoglycan will result the release of the content inside the cell and the death of the cell. In addition to inhibiting DNases, the EDTA in the lysis solution aids the lysozyme to access the peptidoglycan by removing the Mg2+ from the lipopolysaccharide...
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...DNA, one of the most important molecules of all living organisms, holds the genetic imformation that determines the strucure of proteins of every individual organism. Being used in millions of organisms, those living and non-living, DNA can be formed in almost an infinite amount of combinations, making millions of unique traits. DNA has been used in the organisms living on the Earth since the beginning of life, about three or four billion years ago. The search for the chemical identitiy of genetic material started more than a century ago. Im 1869, a method was publshed by Swiss scientist Frederick Miescher on how to separate the cell nuclei from the cytoplasm. In doing that, an acid material was extracted from the cell nuclei which Miescher called "nuclein". Later on, nuclein, which was now called nucleic acid, was found to be involved with different proteins formed in combinations called nucleoproteins. Each nucleotide is made up of a sugar moiety, a phosphate group, and either a purine base or a pyrimidine base. The sugars that had five carbons was given the name ribose whlie the sugar lacking on oxygen atom was givn the name dioxiribose. Since no nucleic acid can contain both of these sugar, there are two types of nucleic acid: ribonucleic acid (RNA), and deoxiribose nucleic acid (DNA). In the 1950, the structure of DNA was cleared with the help from chromatographic separation and X-ray studies. From that it was established that DNA generally is in the form as a duplex...
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...Gene Marker Identification Targeting Toll-Like Receptor 4 (TLR4), Breast Cancer 1 (BRCA1), and Adenosine Triphosphatase 1 Alpha 1 (ATP1A1) Genes: Assessing Their Association With Subclinical Mastitis Cases in Dairy Water Buffaloes, Bubalus bubalis Thesis Proposal Cyndi Candelaria Biendima Patricia Malapit Cabatit Submitted to the Department of Biology College of Arts and Sciences University of the Philippines Manila Padre Faura, Ermita, Manila In partial fulfilment of the requirements for Undergraduate Thesis (BIO 200) TABLE OF CONTENTS Title Page1 Table of Contents2 Introduction3 Review of Related Literature6 Proposed Methodology14 Presentation of Results20 Literature Cited22 Line Item Budget26 Project Timeline27 1.0 INTRODUCTION 1.1 Background of the Study Cases of intramammary infections such as mastitis in water buffaloes contribute to large annual losses in milk production and net profit for smallholder farmers in the Philippines. Social and economic factors might prevent households from diagnosing, treating, and eliminating from circulation those animals or animal products, such as milk, that are afflicted with mastitis or which came from individuals afflicted with mastitis; this is especially true in the case of the asymptomatic subclinical mastitis, which tends to become chronic and difficult to eradicate by conventional antimicrobial therapies (Brouillette & Malouin, 2005; Ng et al., 2010). With the advent of technology...
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...Since the discovery of genetic modification, scientists have been finding new ways to incorporate this process of genetic engineering into applications that could help or cure illnesses and deficiency (The quality or state of being defective or of lacking some necessary quality or element). A well known example of this is the golden rice, the golden rice is a new transformed rice which has been made by genetically modifying the DNA and incorporating the genes of two different rice to make a rice which has the presence of Beta carotene which is the source of vitamin A. This rice has been known to help in the aid of vitamin deficiency. In South and south east Asia vitamin A deficiency is very severe (Check figure 2) with the use of this rice in place we can help these countries consume more Vitamin A. By growing food that is more nutritious and beneficial for our bodies we can essentially create a healthier...
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