...DNA Introduction Large pieces of DNA can be broken up into smaller fragments by bacterial enzymes known as restriction endonucleases (RE). They bind to specific nucleotide sequences within DNA, known as recognition sequences/sites and cleave the phosphate backbone at a point within this sequence. ------------------------------------------------- The recognition sequences are usually fairly short (4-8 base pairs) and have two characteristics: 1) They are PALINDROMIC i.e. the sequence on one of the DNA strands is repeated in reverse on the other e.g. 5'...GAATTC...3' 3'...CTTAAG...5' | 2) They are cleaved by REs in one of two ways, either producing blunt ended fragments, or overhangingfragments | | Since there are only 4 bases in DNA, the probability that a particular cut site is found in a random piece of DNA can be quite high. This is particularly true if the site contains only two bases e.g C and G and the DNA has a higher number of G/C pairs than A/T pairs. In this situation more fragments will be produced. Plasmids are circular pieces of DNA found in bacteria and are frequently used in laboratories to house "foreign" genes. The ability to selectively cut plasmids with REs is extremely useful in genetic engineering. The aim of this experiment is to digest two plasmids, one normal (plasmid 1), one mutant (plasmid 2) and analyse the results to determine the nature of the mutation. Digesting plasmid DNA with restriction...
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...Andrei Vasiliev 01040632 PTC Genotype Determination basing on DNA Samples (Obtained From Individuals with known PTC Test result) that are incubated with Restriction Enzyme (Hae III) Abstract: The ability to taste the bitter compound phenylthiocarbamide (PTC) and related chemicals is bimodal, and all human populations tested to date contain some people who can and some people who cannot taste PTC. Why this trait has been maintained in the population is uncertain but this polymorphism may influence food selection, nutritional status or thyroid metabolism. The gene product that gives rise to this phenotype is unknown, and its characterization would provide insight into the mechanism of bitter taste perception. Although this trait is often considered a simple Mendelian trait, i.e. one gene two alleles, a recent linkage study found a major locus on chromosome 5p15 and evidence for an additional locus on chromosome 7. The development of methods to identify these genes will provide a good stepping-stone between single-gene disorders and polygenic trait. [5. Guo SW, Reed DR.Department of Pediatrics, Medical College of Wisconsin, Milwaukee, USA.] Introduction:The genetic taste phenomenon of PTC was discovered in 1931 when a DuPont chemist named Arthur Fox accidentally released a cloud of a fine crystalline PTC. Standing by his side colleague complained about the bitter taste, while Dr. Fox, who was closer and should have uptaken a strong dose, tasted nothing. Fox then continued...
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...Agarose Gel Electrophoresis for the Isolation of Onion DNA. Abstract Agarose Gel Electrophoresis method was applied on an onion sample to isolate onion DNA. The objective of this experiment was to be able to extract the DNA of an onion in the presence of Ethylenediaminetetraacetic acid. The A260A280 absorbance ratios of isolated DNA were around 1.0593, suggesting that the DNA was not pure and could not be used for further analysis. Moreover, the A260A280 values were lower than 1.8, suggesting that there was a contamination by proteins. The DNA isolated by this protocol was not of sufficient quality. The integrity of the It was concluded that the DNA was not pure due to protein and aromatic contaminations. Introduction Nucleotides play an important role in the cell, either as energy carriers, as coenzymes or as building blocks for nucleic acids. Nucleotides consist of purine bases, adenine ad guanine and pyrimidine bases, cytosine, uracil, or thymine. (Wink, 2006). Deoxyribosenucleic acid is the cells master repository for genetic information. It is a negatively charged, macromolecule consisting of two strands of linked nucleotides, each of which are made up of a deoxyribose sugar residue, a phosphate group and one of bases; thymine, adenine, cytosine or guanine. (Voet and Voet, 2005). It is found coiled up around basic proteins called histones. (Bettelheim and Landesberg, 2009). DNA does not exist as a free molecule in a cell, but instead is associated with proteins and...
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...Introduction: The purpose of this experiment was to successfully transform pGLO plasmid into E.Coli cells. In the first segment of this laboratory exercise, one had to carry out a restriction digest. Restriction digestion is the process of cutting DNA molecules into smaller pieces with special enzymes such as, BamH1 and EcoR1. One had to determine which of the two plasmids, A or B, were pGLO or pWEB. A plasmid is a small circular DNA strand in the cytoplasm of a bacterium (Isite, 2013). In order to determine that, one had to use BamH1 and EcoR1 on two tubes each one with plasmid A and the other with plasmid B to observe the cuts made by each enzyme. The hypothesis for this segment of the laboratory exercise states that pGLO was plasmid B, and pWEB was plasmid A. PGLO is a plasmid which contains green fluorescent protein (GFP). GFP is found in the Aquarius Victoria jelly fish. These jelly fish have a bioluminescent protein that emits blue light. GFP converts the blue light into green light, and that is why these jelly fish emit green light. The pGLO was inserted into E.coli by using transformation. Transformation is the process of transferring genetic material between microbial cells (Tu 2008). Bacterial cells need to be in a state of competency prior to transformation (Isite 2013). Some bacteria naturally achieve this stage when nutrients and oxygen are low (Isite 2013). In the laboratory, bacteria were artificially induced with calcium chloride to be competent for transformation...
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...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 to the positive terminal. During electrophoresis, the gel is submersed in a chamber...
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...Gel Electrophoresis Protocol Materials: 40 ml of 1% agarose gel 40 ml of TBE 1x buffer 4 microL of Ethidium Bromide 50 ml graduated cylinder 250 ml Erlenmeyer flask Ladder *necessary aliquots Gel Cast and comb Mini sub cell chamber Goggles Heat protecting gloves Procedure: 1. Weigh 0.40 g agarose gel 2. Obtain 40 ml of TBE 1x buffer into a 50 ml graduated cylinder 3. Mix the 0.40 agarose gel and the 40 ml TBE 1x buffer into a 250 ml Erlenmeyer flask 4. Cover Erlenmeyer flask with kin wipe 5. Microwave until the mix bubbles vigorously at about 30-40 seconds 6. Remove mix from microwave using heat protecting gloves and allow to cool down for about 5 minutes 7. Sterilize the gel cast with 70% ethanol and balance the cast accordingly 8. Add 4 microL of ethidium bromide into the mix then swirl 9. Pour the mix into the gel cast (pop air bubbles with a pipettor tip) 10. Sterilize comb with ethanol and put it in the gel cast 11. Cover the gel with kin wipe and wait for 30 minutes (if in a hurry put the apparatus in the fridge for 15 mins) 12. Remove the comb slowly and gently 13. Remove the gel and transfer it into a mini sub cell chamber with the wells on the black side 14. Pipet the ladder into the farthest well then the rest of the aliquots accordingly 15. Cover the mini sub cell, attach the wires and change the machine setting to 100volts 16. Run machine for 1 hour 17. Remove gel from the...
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...Agarose Gel Electrophoresis for the Separation of DNA Fragments Pei Yun Lee, John Costumbrado, Chih-Yuan Hsu, Yong Hoon Kim Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles Video Article Chapters 0:05 Title 1:31 Preparation of the Gel 3:21 Setting up Gel Apparatus and Separating DNA Fragments 4:55 Observing Separated DNA Fragments 5:43 Results: Agarose Gel Electrophoresis of PCR Products 6:23 Conclusion Cite this Article Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. Agarose Gel Electrophoresis for the Separation of DNA Fragments. J. Vis. Exp. (62), e3923, doi:10.3791/3923 (2012). Abstract Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule...
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...10 Technology used by Medical Technologist 1. Description: A centrifuge is a piece of equipment that puts an object in rotation around a fixed axis (spins it in a circle), applying a potentially strong force perpendicular to the axis of spin (outward). The centrifuge works using the sedimentation principle, where the centripetal acceleration causes denser substances and particles to move outward in the radial direction. At the same time, objects that are less dense are displaced and move to the center. In a laboratory centrifuge that uses sample tubes, the radial acceleration causes denser particles to settle to the bottom of the tube, while low-density substances rise to the top.[1] There are 3 types of centrifuge designed for different applications. Industrial scale centrifuges are commonly used in manufacturing and waste processing to sediment suspended solids, or to separate immiscible liquids. An example is the cream separator found in dairies. Very high speed centrifuges and ultracentrifuges able to provide very high accelerations can separate fine particles down to the nano-scale, and molecules of different masses. Large centrifuges are used to simulate high gravity or acceleration environments (for example, high-G training for test pilots). Medium-sized centrifuges are used in washing machines and at some swimming pools to wring water out of fabrics. Gas centrifuges are used for isotope separation, such as to enrich nuclear fuelfor fissile isotopes. From: https://en...
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...norfolkhornbreeders.co.uk/history.php I Protocol To perform a quick screen using agarose gel electrophoresis, you collected some cells from the lambs and the potential parents in order to extract and process the DNA ready for electrophoresis. You labeled your tubes as followed: Sample A: Ladder Sample B: Suffolk ewe Sample C: Lamb 1 Sample D: Lamb 2 Sample E: Norfolk Horn ram Sample F: Southdown ram II DNA electrophoresis procedure Read the procedure carefully before you start. well | 1 | 2 | 3 | 4 | 5 | 6 | Sample | A | B | C | D | E | F | Use a plastic pipette to pour some buffer near the comb. GENTLY remove the comb and drop some more buffer in the wells. Using a micropipette, load 20l of each of the samples into separate wells in the agarose gel as indicated in the above table. Do not stab the pipette tip into the wells and pierce the bottom of the gel! When all samples have been loaded onto the gels, place them into the tank. Call a demonstrator to assist you with covering the gels with the buffer. Cover the tank with the lead and connect the electrodes to the electrophoresis power pack the correct way! DNA will migrate towards the positive electrode, which is usually coloured red. Press the RUN button. The gels will be run at 150 volts for about 30 minutes. TURN OFF THE ELECTROPHORESIS POWER PACK. Switch off at...
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...______________________________________ 02/04/16 Date Completed: _________________ BIO195 BIO196 Total score 02/11/16 Date submitted: _________________ T1 Course section: ________ Introduction to Gel Electrophoresis Investigation Title: _________________________________________________ ABSTRACT Your abstract must include the major objective(s) for this investigation along with a summary of what you learned or concluded from your results. Your abstract should be in paragraph form. Gel Electrophoresis is a process by which macromolecules like DNA are separated based on their size. DNA is carried with electricity in an apparatus across an agarose gel matrix. The gel slab is burrowed in a conductive solution, allowing DNA to be measured for analytical and diagnostic purposes. We analyzed DNA from a crime scene and compared it to DNA from five suspects. We trimmed each suspects DNA with restrictive enzyme and then pulsed the tubes in the centrifuge that were then incubated. We then deployed micro amounts of DNA from each suspect into dedicated lanes on the agarose gel slab via micropipets. We introduced an electrical field, the current caused the negatively charged DNA to migrate to the anode pole of the apparatus. While the DNA was migrating to the anode pole, the agarose gel acted like a selectively permeable membrane, giving way to a plurality of migrations for each suspect, inversely proportional to each suspects nucleotide allotment. It came as no surprise that ...
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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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...Hope Hayman DNA Bar Coding: Is Convenient and Accurate for Taxonomy Studies Introduction— Different samples of “wild card” subjects and given subjects (Drosophila melanogaster and the Coquina clams) genomic DNA were acquired and isolated through several steps to amplify the cytochrome c oxidase 1 (CO1) genes. Through comparisons of these wild cards and given subjects mitochondrial CO1 genes with the BLAST library, it was revealed that DNA bar coding is convenient and accurate for taxonomy study. DNA bar coding utilizes the amplification and purification of a specific region of the mitochondrial genome by polymerase chain reactions (PCR). DNA bar coding then uses the PCR products ran in gel electrophoresis to analyze. Materials and Methods— DNA Isolation The wild card specimens were obtained and brought to the lab for DNA isolation. The first step in DNA isolation was to lyse the specimen. The specimens were first homogenized individually in 1.5 mL microtubes. 20 µL of proteinase K was added to each homogenized sample. 200 µL of AL buffer was then added to each sample. The samples were vertexed and incubated at 70oC for 10 minutes. After the incubation, 200 µL of 100% ethanol was added and vortexed again. The next step in DNA isolation is to bind the DNA. The lysate was transferred to a spin column and centrifuged at 8000 rpm for 1 minute, and the flow through was discarded. 500 µL of AW1 buffer was added, then centrifuged at 8000 rpm for 1 minute, and the flow through was...
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...Figure 1: Agarose Gel Electrophoresis depicting the PCR product and genomic DNA of fruit flies This agarose gel image was used to visualized the DNA sequence that was isolated from the fruit flies. Agarose Gel Electrophoresis results from Section 3 Bench 5. EGFR was used as the primer RTK gene for this set and sequence. This is the PCR product. Lane 1, ladder; Lane 2, Genomic DNA and it contains the long streak of DNA; Lane 3, Uncut Plasmid; Lane 4, Linear Plasmid that was digested by EcoRI at 3kb; Lane 5, PCR 1; Lane 6, PCR 2 and these lanes (4-6) 9 show how the specific inserts were applied during PCR; Lane 7, Combined PCR that was purified during miniprep; Our target insert was shown in these lanes at 1 kb too. Lane 8, Ladder. This figure shows...
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...LATEST BIOCHEMICAL TECHNIQUES CAPILLARY ELECTROPHORESIS Capillary electrophoresis is an analytical technique that separates ions based on their electrophoretic mobility with the use of an applied voltage. The electrophoretic mobility is dependent upon the charge of the molecule, the viscosity, and the atom’s radius. In conventional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an electric field. The rate at which the particles moves is directly proportional to the applied electric field; the greater the field strength, the faster the mobility. If two ions are the same size, the greater charge will move the fastest. For ions of the same charge, the smaller particle has less friction and overall faster migration rate. The technique of capillary electrophoresis was designed to separate species based on their size to charge ratio in the interior of a small capillary filled with an electrolyte. Capillary electrophoresis is used most predominately because it gives faster results and provides high resolution separation. It is a useful technique because there is a large range of detection methods available. PRINCIPLE Electrophoresis is the process whereby the movement of ions is produced under the influence of an applied voltage across a field that the ions exist. In electrophoresis, ions that are negatively charged will move or migrate towards the positively charged electrode while ions that are positively charged will...
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...I. 진단혈액 진단혈액 수련항목 (1) : 혈액학적검사 기본 술기 표준수련기간 : 1주 수련내용 : ◆ 용어정의 : • 혈액학적검사 : 혈액세포와 응고에 관련된 일련의 검사를 의미한다. 혈구의 체내분포, 구조, 기능에 관련된 검사, 골수에 분포하는 전구세포에 관한 검사, 혈구에 영향을 끼칠 수 있는 혈장 인자에 관한 검사 및 유전자 이상에 관련된 검사 등을 포괄적으로 포함한다. • 망상적혈구수 : 적혈구 성숙 단계 중 정염색성 적아구(orthochromatophilic normoblast) 바로 다음 단계의 세포로 핵이 빠져나간 직후부터를 의미한다. 미토콘드리아, 중심소체(centriole), 리보솜 등을 함유하고 있으며 말초혈액에서 24-48시간의 성숙과정을 거쳐서 성숙한 적혈구로 된다 (Ref. Williams 16th p373-374) ◆ 숙지할 필수 지식 : • 혈액학 검사에 사용되는 검체와 항응고제의 작용기전 및 종류 • 모세관 혈액의 채취 방법과 용도, 채취 시 주의점 및 정맥혈과의 차이점 • 적혈구침강계수(ESR) 검사의 원리 ◆ 습득할 필수 술기 : • Neubauer chamber의 사용 • 미량법(micromethod)를 이용한 헤마토크리트의 측정 • 수기법을 이용한 망상적혈구수 검사 ◆ 국내외 장비 및 시약 현황 : 해당없음 ◆ 추천되는 참고자료 : • 대한혈액학회. 혈액학, 2006. • 대한진단검사의학회 편, 진단검사의학 제 3판, 2001. • Henry, JB. Clinical Diagnosis and Management by Laboratory Methods, 24th ed. 2006. 보고서 제출 일자 : 200 년 월 일 평가자 : 지도전문의 인 (일자 : 200 년 월 일) 과장 인 (일자 : 200 년 월 일) 수련위원 인 (일자 : 200 년 월 일) 진단혈액 수련항목 (2) : 자동 혈구계산기 표준수련기간 : 2주 수련내용 : ◆ 용어정의 : • 헤마토크릿(Hct) : 혈액 전체 부피에 대한 적혈구 부피의 비율, 단위는 % 또는 L/L • 평균적혈구용적(MCV) : 적혈구의 평균 용적, 단위는 fL, • MCV = Hct (L/L) X 1,000/RBC count (X1012/L) • 평균적혈구혈색소(MCH) : 적혈구 한 개당 혈색소 양, 단위는 pg, • MCH = hemoglobin (g/L)/RBC count (X1012/L) • 평균적혈구혈색소농도(MCHC) : 적혈구 한 개당 평균 혈색소 농도, • 단위는 g/L, MCHC = hemoglobin (g/L)/Hct (L/L) • 적혈구분포지수(RDW)...
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