...BIOL2103 Biological Sciences Laboratory Course Practical 3 Laboratory manual Isolation of nucleic acid and spectrophotometry Introduction: The ability to isolate and quantify nucleic acids accurately and rapidly is a prerequisite for many of the methods used in biochemistry and molecular biology. The concentration of DNA or RNA in a sample, and its condition, are often estimated by running the sample on an agarose gel. Such concentration estimates are semiquantitative at best and are time-consuming. For a more accurate determination of the concentration of DNA or RNA in a sample, a UV spectrophotometer is commonly used. Spectrophotometry uses the fact that there is a relationship between the absorption of ultraviolet light by DNA/RNA and its concentration in a sample. The absorption maximum of DNA/RNA is approx 260nm. The purity of a solution of DNA can be determined using a comparison of the optical density values of the solution at various wavelengths. For pure DNA, the observed A260/A280 ratio will be near 1.8. Elevated ratios usually indicate the presence of RNA. The A260/A280 ratio is used to assess RNA purity. An A260/A280 ratio of 1.8-2.1 is indicative of highly purified RNA. The 260/280 ratio below 1.8 often signal the presence of a contaminating protein or phenol. Alternatively, protein or phenol contamination is indicated by 230/260 ratios greater than 0.5. Workflow Time 2 days before the lab session During lab session 1:30 pm Task Cell...
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...g A New Molecule of Life Life as we know it is far more complex than one can imagine. The smallest molecule in human body can play a large role in determining the genetic outcome or the overall well being of a person. In Peter Nielsen’s “Designing a New Molecule of Life”, he speaks of a molecule that hopefully one day will create a scientific and medical breakthrough. In this essay you will read a summary of Peter Nielsen’s article and the research he has done with this molecule. Peter Nielson, along with many other scientists, have spent years creating and experimenting with a synthetic molecule called peptide nucleic acid (PNA). PNA is an artificial polymer that has many similarities to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It has the same storing features as DNA and RNA while being built on a protein based backbone therefore making it sturdier and simpler than the sugar phosphate-backbone. The molecule was created in hopes of having an immediate affect by pursuing a drug that would target DNA’s composing specific genes, to either enhance or block the gene’s expression. This new drug would be in efforts to interfere with the production of disease producing proteins. Although this molecule has produced highly anticipated medical research, it has also lead to speculations of being the origins of life. In his years of research, Peter Nielsen and his colleagues wanted to achieve the ability of PNA recognizing double-stranded or duplex DNA having specific...
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..._ However, the helix structure (to be more specificly, the glycosidic bonds) is easily wrecked by heat (boiling temperature of water) and acid treatment (HCl). Once the helix structure of homopolycarbohydrate is broken, the conformation with iodine no longer exists. It leads to the gradual loss of blue color. | 2 | | | 4 | | | 6 | | | 8 | | | 10 | | | | No color change | | Color of Lugol | | | 2.PROTEINS 2.2.2. Task-Qualitative Detection of Proteins Protein solution | Original color | After 10% NaOH | After 0.5% CuSO4 | 1. Egg albumin | Colorless | Colorless | Blue->purple | 2. Fresh cow milk | White | White | Blue->purple | Explanation: NaOH doesn’t react with Proteins, so there’s no change to the protein solutions Next, when CuSO4 is added, it reacts with NaOH to form Cu(OH)2 (blue) first. 2NaOH+ CuSO4 -> Cu(OH)2(s) + Na2SO4 Then Cu(OH)2 and Proteins together form the complex, which gives out the purple color. This reaction (called biuret reaction) is useful to detect the protein presence. (write down the chemical equation, GOOGLE) 3. LIPIDS 4. NUCLEIC ACIDS 4.1.General Introduction Nucleic acids are large biological molecules essential for all known forms of life. They include DNA (deoxyribonucleic acid) and RNA(ribonucleic acid). Together with proteins, nucleic acids are the most important biological macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting...
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...What biological principle is illustrated by the Heike crabs? Selection by humans Sagan says that if artificial selection can produce vast changes in a short period of time, then what must nature be able to do, given the age of the ear (4.5 billion years or so)? Don't just quote the video here. Explain what he means! Nature must be able to produce and create. What are the basic “steps” in natural selection as Sagan describes them? What questions does he raise for you? Natural Selection happens when human’s changes have created other changes without humans directly deciding to do so. Sagan describes the basic steps as there are more creatures then can survive. Less adapted have a less chance of surviving and producing off spring. Sudden changes in heredity can be pushed on to the off springs. Environmental changes can have an impact on what mutations will help survival. Thus, slow changes produce new species. Natural Selection was discovered by Charles Darwin and Alfred Russel Wallace. Explain the Watchmaker Hypothesis as an argument against natural selection. How does Sagan address it? “Our ancestors looked at the intricacy and the beauty of life and saw evidence for a great designer. The simplest organism is a far more complex machine than the finest pocket watch. And yet, pocket watches don’t spontaneously self-assemble or evolve in slow stages on their own from say, grandfather clocks”. (Sagan, C) It’s impossible to just look at something and understand...
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...Title of Experiment 9: Measurement of Nucleic Acid Solutions Objectives: 1. To study the principle of gel electrophoresis. 2. To use gel electrophoresis method to measure the nucleic acid solutions. 3. To learn the technique of nuclei acid measurement by using gel electrophoresis. Introduction: Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Most every molecular biology research laboratory routinely uses agarose gel electrophoresis for the preparation and analysis of DNA. The components of gel electrophoresis system is power supply and a chamber, agarose gel which is a porous material that allows molecules migrate through, buffer with a mixture of water and ions, and gel casting tray and comb. Electrophoresis is a method of separating substances based on the rate of movement while under the influence of an electric field. Agarose is a polysaccharide purified from seaweed. An agarose gel is created by suspending dry agarose in a buffer solution, boiling until the solution becomes clear, and then pouring it into a casting tray and allowing it to cool. The result is a flexible gelatin-like slab. Nucleic acids are composed of chains of nucleotides. The ‘backbone’ of the nucleic acid structure is a repeating chain of phosphate groups and pentose sugars. At certain pH values, the oxygen atoms in the phosphate groups ionize, giving the molecule an overall negative charge. If exposed to an electric field, these molecules are attracted...
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...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 (LPS) layer, which will disrupt the membrane of the bacteria (1). The isolation step requires the removal of other macromolecules, such as protein and RNA, in the lysate. Protein can be dissociated from nucleic acids with phenol and chloroform. Proteolytic enzymes including pronase and proteinase K are often added to further remove protein. RNA, on the other hand, can be removed by RNase. Lastly, the DNA can be precipitated out from alcohol. In order for the nucleic acid to precipitate out of alcohol, some sort of cations, such as sodium and ammonium, are used to shield the negative charge of nucleic acid. As a result, nucleic acid will have a reduced solubility in solution and become insoluble pellet. The DNA extraction procedure provides a sample of purified DNA extract that can be used for restriction enzyme digest in upcoming experiment. Plasmid isolation protocol (mini-prep) is somewhat like genomic DNA isolation but with some variation to the content of lysis buffer, method of macromolecule removal and DNA collection. The focus of mini-prep is that plasmid isolation requires the separation from not only the cellular components but also from the genomic...
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...CHAPTER 9: NUCLEIC ACIDS 9.1 Levels of Structure in Nucleic Acids Primary (1o) Structure – order of bases in the polynucleotide sequence Secondary (2o) Structure – 3D conformation of backbone Tertiary (3o) Structure – supercoiling of the molecule Quaternary (4o) Structure – interaction between DNA & proteins Two principle types of nucleic acids - DNA (deoxyribonucleic acid) - RNA (ribonucleic acid) How do DNA and RNA differ? Ribosomes: polypeptide-generating machinery of the cell Tobacco mosaic virus: nucleic acid strand winds through a cylinder of coat-protein subunits 9.2 The Covalent Structure of Polynucleotides Nucleotides: monomers of nucleic acids 1. Nitrogenous base 2. Sugar 3. Phosphoric acid residue Order of nucleic acids of DNA contains the information necessary to produce the correct amino acid sequence in the cell’s proteins What are the structures and components of the nucleotides? Nucleic acid bases (nucleobases): one or two-ring nitrogenous aromatic compound - Pyrimidines – single-ring aromatic compounds Cytosine – DNA & RNA Thymine – substitute for Uracil in DNA (sometimes in RNA) Uracil – RNA only - Purines – double-ring aromatic compounds Adenine – DNA & RNA Guanine – DNA & RNA Methylation can modify bases Nucleoside - base + sugar covalently bonded - lacks phosphate group - base forms a glycosidic linkage with sugar Ribonucleoside: β-D-ribose Deoxyribonucleoside: β-D-deoxyribose The glycosidic...
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...Gel electrophoresis Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge and/or size (IEF agarose, essentially size independent) and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.[1] Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through an agarose matrix. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving.[2] Proteins are separated by charge in agarose because the pores of the gel are too large to sieve proteins. Gel electrophoresis can also be used for separation of nanoparticles. Gel electrophoresis uses a gel as an anticonvective medium and/or sieving medium during electrophoresis, the movement of a charged particle in an electrical field. Gels suppress the thermal convection caused by application of the electric field, and can also act as a sieving medium, retarding the passage of molecules; gels can also simply serve to maintain the finished separation, so that a post electrophoresis stain can be applied.[3] DNA Gel electrophoresis is usually performed for analytical purposes, often after...
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...D38–D51 Nucleic Acids Research, 2011, Vol. 39, Database issue doi:10.1093/nar/gkq1172 Published online 20 November 2010 Database resources of the National Center for Biotechnology Information Eric W. Sayers1,*, Tanya Barrett1, Dennis A. Benson1, Evan Bolton1, Stephen H. Bryant1, Kathi Canese1, Vyacheslav Chetvernin1, Deanna M. Church1, Michael DiCuccio1, Scott Federhen1, Michael Feolo1, Ian M. Fingerman1, Lewis Y. Geer1, Wolfgang Helmberg2, Yuri Kapustin1, David Landsman1, David J. Lipman1, Zhiyong Lu1, Thomas L. Madden1, Tom Madej1, Donna R. Maglott1, Aron Marchler-Bauer1, Vadim Miller1, Ilene Mizrachi1, James Ostell1, Anna Panchenko1, Lon Phan1, Kim D. Pruitt1, Gregory D. Schuler1, Edwin Sequeira1, Stephen T. Sherry1, Martin Shumway1, Karl Sirotkin1, Douglas Slotta1, Alexandre Souvorov1, Grigory Starchenko1, Tatiana A. Tatusova1, Lukas Wagner1, Yanli Wang1, W. John Wilbur1, Eugene Yaschenko1 and Jian Ye1 1 Downloaded from http://nar.oxfordjournals.org/ by guest on March 20, 2015 National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Building 38A, 8600 Rockville Pike, Bethesda, MD 20894, USA and 2University Clinic of Blood Group Serology and Transfusion Medicine, Medical University of Graz, Auenbruggerplatz 3, A-8036 Graz, Austria Received September 16, 2010; Revised October 29, 2010; Accepted November 1, 2010 ABSTRACT In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology...
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...Bio Lab Practical #1 Solute- dissolved in solvent Solvent- a substance in which another substance is dissolved Solution- a liquid mixture in which the solute and solvent are uniformly distribute Molecular weight- average mass of a molecule Mole- a measurement of quantity or numbers pH- the measurement of acidity or basicity in an aqueous solution Prokaryotic- single-celled organism that lacks a membrane-bound nucleus, mitochondria, or any membrane-bound organelles Eukaryotic- has true nucleus, nuclear pores, and organelles Organelles- a specialized part of a cell having specific fucntions, cell organ Diffusion- means of passive transport, three types: facilitated, simple, and channel Osmosis- the movement of water from high potential to low potential Brownian movement- random movement of microscopic particles in liquid Dialysis- the separation of particles in a liquid on the basis of differences in their ability to pass through a membrane Dialysis tubing- semi-permeable membrane tubing Biologically Important Molecules: Carbohydrates, proteins, lipids, and nucleic acids – most organic compounds in living organisms Dehydration- removal of water molecule and covalently bonding two subunits Hydrolysis- breaking bonds in subunits by adding water Positive control- contains the variable for which you are test. It reacts positively and demonstrates the test’s ability to detect what you expect Negative control- does not contain...
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...Pre-AP Biology Chapter 2 Test Chemistry of Life Multiple Choice (1 point each) Identify the choice that best completes the statement or answers the question. ____b 1. The space surrounding the nucleus of an atom contains |a. |protons. |c. |neutrons. | |b. |electrons. |d. |ions. | ____c 2. If an atom contains 3 protons, 4 neutrons, and 3 electrons, its mass number would be |a. |3. |c. |7. | |b. |4. |d. |11. | c____ 3. Isotopes are atoms of the same element with the same number of protons and |a. |a different number of |c. |a different number of neutrons.| | |electrons. | | | |b. |a different number of |d. |the same number of neutrons. | | |molecules. | | | ___d_ 4. Which of the following is...
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...solve human issues including overcoming diseases and physical handicaps. Biotechnology has made breakthroughs in various nucleic acid and protein studies, and it has given scientists the ability to comprehend the complicated construction of DNA. Understanding the blueprints of DNA and how it is translated into proteins gives chemists the opportunity to define features like a genome, which is an organism’s complete set of DNA. Our DNA is important because it is our hereditary code, and that is what controls our appearance, behavior, and tendencies....
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...579 Atomic force microscopy and other scanning probe microscopies Helen G Hansma and Lía Pietrasanta The highlight of the past year is the unfolding and refolding of the muscle protein titin in the atomic force microscope. A related highlight in the intersection between experiment and theory is a recent review of the effects of molecular forces on biochemical kinetics. Other advances in scanning probe microscopy include entropic brushes, molecular sandwiches and applications of atomic force microscopy to gene therapy. Address Department of Physics, University of California, Santa Barbara, CA 93106, USA Current Opinion in Chemical Biology 1998, 2:579–584 http://biomednet.com/elecref/1367593100200579 © Current Biology Ltd ISSN 1367-5931 Abbreviations AFM atomic force microscopy/microscope SFM scanning force microscopy/microscope SICM scanning ion conductance microscopy/microscope SPM scanning probe microscopy/microscope STM scanning tunneling microscopy/microscope A new journal, Probe Microscopy, was launched in 1997 as a forum specifically devoted to the science and technology of SPM. AFM and SFM have been also newsworthy items in Science and Nature in the past year [14••,15•–17•,18••,19]. An introduction to AFM is covered well in a recent issue of Current Opinion in Chemical Biology, which describes and illustrates the design and mode of operation of AFM [4••]. The AFM images sample surfaces by raster-scanning a sharp tip back and forth over the surface. The tip is on...
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...Biosensors and Bioelectronics 25 (2009) 661–667 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Review Detection of microorganisms using biosensors—A smarter way towards detection techniques Madhura Nayak 1 , Akhil Kotian 1 , Sandhya Marathe 1 , Dipshikha Chakravortty ∗ Centre for Infectious Disease Research and Biosafety Laboratories, Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India a r t i c l e i n f o a b s t r a c t Along with useful microorganisms, there are some that cause potential damage to the animals and plants. Detection and identification of these harmful organisms in a cost and time effective way is a challenge for the researchers. The future of detection methods for microorganisms shall be guided by biosensor, which has already contributed enormously in sensing and detection technology. Here, we aim to review the use of various biosensors, developed by integrating the biological and physicochemical/mechanical properties (of tranducers), which can have enormous implication in healthcare, food, agriculture and biodefence. We have also highlighted the ways to improve the functioning of the biosensor. © 2009 Elsevier B.V. All rights reserved. Article history: Received 8 July 2009 Received in revised form 22 August 2009 Accepted 25 August 2009 Available online 31 August 2009 Keywords: Biosensors Microorganisms Biodefence...
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...cellulose Lipids H O H H C H H C H H C O C O O C O O These come in many varieties: fats, oils, cholesterol, steroids, and more, and have uses in cellular membranes, insulating and protecting, and also act as a minor energy supply C H Proteins Proteins have several uses, such as for transport and structure; but they are also the basic components of all enzymes, hormones, antibodies, haemoglobin, ribosomes, and many more materials Water H O H Another essential life component, this is the most important content of many reactions forming most of these molecules, and also metabolic reactions; water is also an essential structural component in plants, and in the diet of animals Nucleic acids These are responsible for the formation of both DNA and all forms of RNA molecules, consisting of individual nucleotides www.asbiology101.wordpress.com Enzymes These are proteins which are used in many reactions – their function is to catalyse metabolic reactions in the vast majority of living organisms There is a lot of chemistry knowledge in the Biological Molecules section of this module, which is why it is important that you are aware of a few chemistry basics, such as the types of chemical bond. This unit on Biological Molecules is centred...
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