Use of Drosophila Melanogaster as a Model System in the Study of Human Sodium- Dependent Multivitamin Transporter. Michael Brinton BIOL 230W.
|
|
- Ginger Nash
- 6 years ago
- Views:
Transcription
1 Use of Drosophila Melanogaster as a Model System in the Study of Human Sodium- Dependent Multivitamin Transporter Michael Brinton BIOL 230W October 2013 TA: Sashi Gollapudi
2 Introduction Many human diseases are caused at the genomic level by mutations in DNA coding. These mutations cause defecting proteins to be built, which can have devastating effects downstream. The genomic aspect to human disease can be difficult to study for many reasons, including the lack of family genomic history (due to lifespan) and limited number of progeny on top of the possible danger for the human subjects. Because of this, researchers have developed model systems in order to study human disease. One of these models is the fruit fly, Drosophila melanogaster. Fortunately, proteins tend to be made up of protein domains. These domains are functional groups of amino acids that tend to retain their function regardless of where they appear. They tend to be highly conserved through evolution due to their specialized function in the cell. Thus, they appear in many organisms. In fact 90% of protein domains are replicated in all eukaryotic organisms. This allows for there to be a great amount of homology between certain organisms. Drosophila and Homo sapiens are two of these organisms. The study of drosophila a homolog has been made easier by the production of cdna libraries. (1) cdna is copy DNA or complimentary DNA. It is a copy of the organism s original DNA, made by reverse transcription of mrna back to DNA. This reaction is catalyzed by reverse transcriptase. cdna is, essentially, a copy of the organism s DNA. However, since the cdna is made from mature mrna, it lacks the introns that may have been present in the original DNA. Thus, cdna only contains coding regions of DNA. This makes genes readily identifiable, and can simplify the study of genes. Collections of cdna can be made and placed into a host cell. This collection is called a cdna library. Together the host cells containing the cdna libraries constitute a portion if the original organism s transcriptome. Creating cdna
3 libraries in cells like bacteria can make replication and collection of the cdna libraries for study much easier. In this experiment, cdna libraries from Drosophila melanogaster will be used within E. coli cells to start. The plasmids containing the cdna will be isolated and the cdna will be sequenced and analyzed. This drosophila cdna will be compared against the human genome to attempt to find a human homolog to the genes of drosophila, thus, showing the importance of drosophila as a model system for human disease. Methods and Materials Taken from: cdna Isolation and Analysis: Identifying potential Drosophila melanogaster models of human disease. Edited by Nelson, K. and Burpee, D. Department of Biology, The Pennsylvania State University, University Park, PA. (2013) Week 1 - Bacteria Culture Selection and Growth: E. coli containing a plasmid with a gene for ampicillin resistance and a Drosophila cdna sequence were grown on an agar plate. From this, two colonies were selected to continue in the process. Each of these colonies was placed in a glass culture tube containing 2 ml of Luria broth plus ampicillin. These cultures were grown overnight at 37 degrees Celsius. After growing, the cultures were stored at 4 degrees C for six days. Week 2 - DNA Isolation: Plasmid DNA was isolated using the QuickLyse Miniprep Plasmid DNA purification system. Cells were removed from the liquid broth and were resuspended in a lysis solution. Cells were lysed and, using a centrifuge for 1 minute at 13,000 rpm, DNA was captured on the membrane of the spin column. This DNA was then washed in an isopropanol buffer and then eluted in a low-salt buffer. This product would be used in the following steps.
4 Week 2 - PCR Setup: A small portion of each plasmid DNA was set up for a PCR reaction. Each PCR tube was filled with 24 µl of the PCR master mix (containing the buffer, dntps, two primers and the Taq polymerase). Added to these tubes was 1 µl of DNA A, DNA B, or sterile water. These tubes were labeled 5A, 5B, and 5N. The contents of these tubes can also be seen in Table 1. Table 1. PCR Tube Contents Tube A Tube B Tube Neg Master Mix 24 µl 24 µl 24 µl Plasmid DNA A 1 µl Plasmid DNA B µl --- Sterile Water µl These were then submitted to the Teaching Assistant for PCR processing. These samples were then stored in the freezer until week 3. Week 3 - Agarose Gel Electrophoresis: The agarose gel was made by mixing 250 mg of agarose with 25 ml of 1XTAE buffer and microwaving for 40 seconds. 1 µl of ethidium bromide was added to this mixture before casting. Once the gel was cooled and solid, the electrophoresis unit was filled with 1XTAE buffer. Once casted, with 8 wells, the following were prepared. 2 µl of each of the Plasmid DNAs were mixed separately with 8 µl sterile water and 2 µl of 6x loading dye (Bromophenol blue and Xylene cyanol). 4 µl of 6x loading dye was added to each of the PCR tubes from the previous week. The wells of the gel were then loaded as follows. Well 1, 5 µl DNA ladder. Well 2, 12 µl Plasmid A DNA. Well 3, 12 µl Plasmid A PCR. Well 4, 12 µl Plasmid B DNA. Well 5, 12 µl Plasmid B PCR. Well 6, PCR Negative Control. Electrophoresis was run at 107 Volts until the dye had migrated approximately halfway across
5 the gel. The gel was removed from the unit and photographed under UV light. After viewing the photograph and confirming the presence of usable DNA, these sampled were submitted for sequencing at the nucleic acid facility on campus. Sequencing was performed using Sanger s dideoxy method, and results from sequencing were reported back in ab1 files. Week 4 - Sequence Analysis: The.ab1 file provided by the sequencing facility was imported into MEGA for analysis. A sequence of approximately 450 base pairs was selected starting around base 70 of the provided sequence data. This selection was used to run a nucleotide Blast search. Accession numbers for the drosophila gene and protein and the human homolog gene and protein were retrieved. These were used in cdart (an NCBI resource) to retrieve information about the protein structure and function. A literature search was run using NCBI PubMed to retrieve more information regarding these proteins. Results When bacteria colonies were observed at the beginning of week two, one could see a pellet at the bottom of the tube. The bacteria that had grown overnight had clumped at the bottom of the tube. After isolation of the DNA and further preparation, the samples were ready for PCR. PCR and agarose gel electrophoresis were performed to acquire the gel in Figure 1.
6 Figure 1. Agarose Gel with Plasmid DNA and PCR In Figure 1, the left-most lane (lane 1) contains the DNA ladder. Lanes 2 and 4 contain Plasmid A and B DNA respectively, and lanes 3 and 5 contain Plasmid A and B PCR respectively. Lane 6 contains the PCR negative control. As one can see, both the DNA and the PCR contain DNA of relatively large size. Also, the negative control shows no DNA, which confirms no contamination of the samples. After confirmation of DNA presence, the Plasmid DNA samples were submitted for sequencing as described above. The Blast search revealed that our sequence was drosophila gene NM_ which codes for drosophila protein NP_ This is homologous to human gene NM_ which codes for human protein NP_ This protein is a sodium-dependent multivitamin transporter. This protein consists of only one domain, SLL5-6-like sbd solute carrier, solute binding domain. This domain is a sodium/glucose transporter. It also functions as a sodium and calcium dependent
7 neurotransmitter transporter. It consists of functional core of 10 transmembrane helices, and functions mainly as a transmembrane transporter. Discussion This experiment ended with the acquisition of information on a drosophila gene and its human homolog. These genes are very similar and code for almost-identical proteins. These proteins contain the same domain. As mentioned above, the gene isolated from the cdna library was a gene to code a sodium-dependent multivitamin transporter. This protein is responsible for the uptake of certain vitamins into the cell. Many research studies, including those cited below, focus on the function of the sodium dependent multivitamin transporter in the uptake of biotin. Research has shown that this protein may play a part in some diseases. For example a study by Patel showed that there was s higher expression of the sodium-dependent multivitamin transporter (SMVT) in breast cancer (T47D) cells than in normal mammary epithelial (MCF- 12A) cells. The SMVT is an important carrier-mediated system in the uptake of biotin into the cells. In this study, they found that the cancer cells were able to uptake more vitamins and more biotin because of the higher expression of this gene. This led the researchers to the possibility of using this gene and/or protein in the study of biotin-conjugated anti-cancer drugs and drug delivery systems. (4) Another study focused on the role of the sodium-dependent multivitamin transporter in the uptake of biotin in three different types of cells. In this study, they confirmed the presence of SMVT and its role in the uptake and transport of biotin and the conjugate in MDCK-MDR1 cells. This study led the researchers to use these MDCK-MDR1 cells in the study of the permeability of biotin conjugated prodrugs. The goal is to study the use of drugs such as HIV
8 protease. This study shows again that the SMVT can potentially be used to make drugs targeting specific cell, in this case cells that cause an otherwise difficult to treat disease. (2) A third study also investigated the role of the sodium-dependent multivitamin transporter to enhance the permeability of some drugs and the targeting of specific cells. In this case, they used human-derived prostate cancer (PC-3) cells. This study also focused on the uptake of biotin in these cells. The study also investigated the regulatory pathways for the uptake of biotin. In the end, the study confirmed the expression of the sodium-dependent multivitamin transporter and its functionality within the PC-3 cells. This again led the researchers to the possibility of using the SMVT as an agent in the treatment of prostate cancer. (3) As one can see, the sodium-dependent multivitamin transporter has the potential to be targeted as the transporter for otherwise poorly-permeable drugs. All of the studies above use some sort of model system. In this experiment, we studied drosophila melanogaster as a potential model system for human disease. The fact that we were able to isolate and sequence drosophila DNA from the plasmids in E. coli shows that drosophila can be potentially used as a model system. Finding a human homologue for the isolated gene that was almost identical to the Drosophila gene confirms that Drosophila could be used as a model system. The studies mentioned above show the importance of using model systems. In none of these studied did they use a human test subject. Instead, they scaled down their experiments by using a model. None of these studies happened to use drosophila as the model; however, this experiment shown the possibility of doing so. The drosophila model could have significance in molecular research. Just as the drosophila SMVT gene was very similar to a human gene, so are many of its other genes. The drosophila model is not limited just to studying the SMVT. It could be used in a wide range of cellular and molecular research.
9 Conclusion Obviously, SMVT plays an integral role within the human, or drosophila, cell. This protein can be targeted for drug delivery by enhancing the permeability of otherwise poorly-permeable drugs. Research into the use of the SMVT can be led using model systems, such as the drosophila system investigated in this experiment. The homology between drosophila and human genes shows that drosophila could be used as a model system for studying human disease from a genomic standpoint.
10 Works Cited 1. cdna Isolation and Analysis: Identifying potential Drosophila melanogaster models of human disease. Edited by Nelson, K. and Burpee, D. Department of Biology, The Pennsylvania State University, University Park, PA. (2013) 2. Luo S, Kansara VS, Zhu X, et al. Functional characterization of sodium-dependent multivitamin transporter in MDCK-MDR1 cells and its utilization as a target for drug delivery. Mol Pharm. 2006;3: Patel, Mitesh (10/2012). "Molecular expression and functional activity of sodium dependent multivitamin transporter in human prostate cancer cells". International journal of pharmaceutics ( ), 436 (1-2), p Vadlapudi, Aswani Dutt (01/2013). "Biotin uptake by T47D breast cancer cells: Functional and molecular evidence of sodium-dependent multivitamin transporter (SMVT)". International journal of pharmaceutics ( ), 441 (1-2), p. 535.
HiPer RT-PCR Teaching Kit
HiPer RT-PCR Teaching Kit Product Code: HTBM024 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 4 hours Agarose Gel Electrophoresis: 45 minutes Storage Instructions: The
More informationBiol/Chem 475 Spring 2007
Biol/Chem 475 Spring 2007 Goal of lab: For most of the quarter, we will be exploring a gene family that was first discovered in fruitlfies and then found to be present in humans and worms and fish and
More informationDNA miniprep by Alkaline Lysis (activity)
DNA miniprep by Alkaline Lysis (activity) Contents 1 Alkaline Lysis 2 Exercise 1: Plasmid DNA Mini-Prep by Alkaline Lysis 3 Identification of Plasmid DNA 4 Exercise 2: Restriction Digestion Identification
More informationHiPer Gel Extraction Teaching Kit (Column Based)
HiPer Gel Extraction Teaching Kit (Column Based) Product Code: HTBM010 Number of experiments that can be performed: 10 Duration of Experiment Agarose Gel Electrophoresis: 1 hour Protocol: 1 hour Agarose
More informationAmgen Laboratory Series. Tabs C and E
Amgen Laboratory Series Tabs C and E Chapter 2A Goals Describe the characteristics of plasmids Explain how plasmids are used in cloning a gene Describe the function of restriction enzymes Explain how to
More informationMolecular Cell Biology - Problem Drill 11: Recombinant DNA
Molecular Cell Biology - Problem Drill 11: Recombinant DNA Question No. 1 of 10 1. Which of the following statements about the sources of DNA used for molecular cloning is correct? Question #1 (A) cdna
More informationApplication of Molecular Biology tools for cloning of a foreign gene
IFM/Kemi Linköpings Universitet September 2013/LGM Labmanual Project course Application of Molecular Biology tools for cloning of a foreign gene Table of contents Introduction... 3 Amplification of a gene
More informationSite-directed mutagenesis of proteins
IFM/Kemi Linköpings Universitet August 2013/LGM Labmanual Site-directed mutagenesis of proteins Figur 1: Flow-chart of the site-directed mutagenesis lab exercise 2 Site-specific mutagenesis Introduction
More informationDNA Visualizer Extraction Kit
DNA Visualizer Extraction Kit Catalog Number D0006 50 reactions Version: 03 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use... 3 Background... 3 General Information...
More informationSOP for Quantitation of the Hepatitis C Virus (HCV) Genome by RT-PCR
Virus Bank SOP-HCV-001 1. Scope SOP for Quantitation of the Hepatitis C Virus (HCV) Genome by RT-PCR 1.1 This procedure describes a method for quantitation of the HCV genome in HCV-infected cells by RT-PCR.
More informationHurricane Miniprep Kit PROTOCOL
Hurricane Miniprep Kit PROTOCOL Description: The Hurricane Miniprep Kit is designed for purification of up to 25 ug of high purity plasmid DNA from a starting volume of 2-5 ml of bacterial culture. The
More informationLinköpings Universitet. Site-directed mutagenesis of proteins
IFM/Kemi August2011/LGM Linköpings Universitet Site-directed mutagenesis of proteins Competent E. coli cells Site-specific mutagenesis Analysis on agarose gel Transformation of plasmids in E. coli Preparation
More informationEZ-10 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK
EZ-0 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK (Bacteria, Plant, Animal, Blood) Version 8 Rev 05/0/03 EZ-0 Genomic DNA Kit Handbook Table of Contents Introduction Limitations of Use Features Applications
More informationPresto Mini Plasmid Kit
Instruction Manual Ver. 03.06.17 For Research Use Only Presto Mini Plasmid Kit PDH004 (4 Preparation Sample Kit) PDH100 (100 Preparation Kit) PDH300 (300 Preparation Kit) Advantages Sample: 1-7 ml of cultured
More informationDescription...1 Components...1 Storage... 1 Technical Information...1 Protocol...2 Examples using the kit...4 Troubleshooting...
QuickClean II Gel Extraction Kit Cat. No. L00418 Technical Manual No. TM0594 Version: 03042011 I II III IV V VI VII VIII Description......1 Components.....1 Storage.... 1 Technical Information....1 Protocol.....2
More informationPlasmid DNA Isolation Column Kit Instruction Manual Catalog No. SA-40012: 50 reactions SA-40011: 100 reactions
Plasmid DNA Isolation Column Kit Instruction Manual Catalog No. SA-40012: 50 reactions SA-40011: 100 reactions Maxim Biotech, Inc. 780 Dubuque Avenue, So. San Francisco, CA 94080, U.S.A. Tel: (800) 989-6296
More informationHiPer Random Amplification of Polymorphic DNA (RAPD) Teaching Kit
HiPer Random Amplification of Polymorphic DNA (RAPD) Teaching Kit Product Code: HTBM031 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 3.5 hours Agarose Gel Electrophoresis:
More information7.1 Techniques for Producing and Analyzing DNA. SBI4U Ms. Ho-Lau
7.1 Techniques for Producing and Analyzing DNA SBI4U Ms. Ho-Lau What is Biotechnology? From Merriam-Webster: the manipulation of living organisms or their components to produce useful usually commercial
More informationHiPer Yeast Genomic DNA Extraction Teaching Kit
HiPer Yeast Genomic DNA Extraction Teaching Kit Product Code: HTBM013 Number of experiments that can be performed: 10 Duration of Experiment: 3 days Day 1: Revival of Host Day 2: Inoculation of culture
More informationUnderstanding the Cellular Mechanism of the Excess Microsporocytes I (EMSI) Gene. Andrew ElBardissi, The Pennsylvania State University
Understanding the Cellular Mechanism of the Excess Microsporocytes I (EMSI) Gene Andrew ElBardissi, The Pennsylvania State University Abstract: Hong Ma, The Pennsylvania State University The Excess Microsporocytes
More informationTaura Syndrome Virus (TSV) RT-PCR Kit
Revision No.: ZJ0001 Issue Date: Aug 28th, 2007 Taura Syndrome Virus (TSV) RT-PCR Kit Cat. No.: AR-0200-03 For use with Conventional PCR Instrument or Real time PCR Instrument User Manual For in vitro
More informationBIO 121 LAB 10 - DNA I
BIO 121 LAB 10 - DNA I All cellular organisms store their hereditary information as the precise sequence of nucleotides in DNA, just as written information is stored as the precise sequence of letters
More informationINDEX INDEX 0 KIT COMPONENTS 1 STORAGE AND STABILITY 1 INTRODUCTION 1 IMPORTANT NOTES 2 EUROGOLD TOTAL RNA ISOLATION PROTOCOL 2 DNA CONTAMINATION 5
INDEX INDEX 0 KIT COMPONENTS 1 STORAGE AND STABILITY 1 INTRODUCTION 1 IMPORTANT NOTES 2 EUROGOLD TOTAL RNA ISOLATION PROTOCOL 2 DNA CONTAMINATION 5 QUANTITATION AND STORAGE OF RNA 5 RNA QUALITY 5 TROUBLESHOOTING
More informationMolecular Techniques Third-year Biology
PLANNING Genetics Lab practices Molecular Techniques. Genetics Lab practices protocol. 2015-16 PCR-DIRECTED MUTAGENESIS, MOLECULAR CLONING AND RESTRICTION ANALYSIS Sessions 1 & 2 (2x3 hours): PCR-directed
More informationEZ-10 SPIN COLUMN HANDBOOK
EZ-0 SPIN COLUMN HANDBOOK EZ-0 Spin Column Plasmid DNA Mini Kit EZ-0 Spin Column PCR Products Purification Kit EZ-0 Spin Column DNA Gel Extraction Kit Version 20.0 Rev 3/23/205 ISO900 Certified 20 Konrad
More informationB. Incorrect! Ligation is also a necessary step for cloning.
Genetics - Problem Drill 15: The Techniques in Molecular Genetics No. 1 of 10 1. Which of the following is not part of the normal process of cloning recombinant DNA in bacteria? (A) Restriction endonuclease
More informationSynthetic Biology for
Synthetic Biology for Plasmids and DNA Digestion Plasmids Plasmids are small DNA molecules that are separate from chromosomal DNA They are most commonly found as double stranded, circular DNA Typical plasmids
More informationITS Sequencing in Millepora. 10/09 Subcloning DNA Fragments into pbluescript Preparation of pbluescript Vector
Page 1 of 5 10/09 Subcloning DNA Fragments into pbluescript Preparation of pbluescript Vector 1. Digest 1 µg of pbluescript with Eco RI 2. Following digestion, add 0.1 volumes of 3M sodium acetate (ph
More informationThe Biotechnology Toolbox
Chapter 15 The Biotechnology Toolbox Cutting and Pasting DNA Cutting DNA Restriction endonuclease or restriction enzymes Cellular protection mechanism for infected foreign DNA Recognition and cutting specific
More informationWhat is DNA. DNA DNA is the genetic material. Is Is a double helix. Made Made up of subunits called nucleotides Nucleotide
What is DNA DNA DNA is the genetic material. Is Is a double helix. Made Made up of subunits called nucleotides Nucleotide made of: 1. hosphate group 2. 5-carbon sugar 3. Nitrogenous base 2 O 4' T 1' to
More informationBring a Molecular Cell Biology Laboratory into the Classroom of HKUST
Bring a Molecular Cell Biology Laboratory into the Classroom of HKUST Prof. Kathy Q. Luo and Prof. Donald C. Chang Dept. of Chemical Engineering, Bioengineering Graduate Program and Dept. of Biology HK
More informationDNA PURIFICATION KITS PLASMID DNA ISOLATION
LIST -NEW v.01-02- DNA PURIFICATION KITS PLASMID DNA ISOLATION Plasmid Mini ultrapure, high and medium copy number plasmid DNA isolation from 1.5-3 ml of bacteria culture PURIFICATION TECHNOLOGY SM 020-50
More informationGel/PCR Extraction Kit
Gel/PCR Extraction Kit Item No: EX-GP200 (200rxns) Content Content Binding Buffer BD Wash Buffer PE Elution Buffer (10 mm Tris-HCl, ph 8.5) Spin Columns EX-GP200 80 ml 20 mlx3 10 ml 200 each Description
More informationNucleic acid-free silica-matrix: Regeneration of DNA binding columns
MAXXBOND ready-to-use - Kit for the regeneration of DNA binding columns with pure silica matrices Product No. MB007 Nucleic acid-free silica-matrix: Regeneration of DNA binding columns efficient and easy
More informationTIANgel Mini DNA Purification Kit
TIANgel Mini DNA Purification Kit For DNA purification from agarose and polyacrylamide gels www.tiangen.com/en DP130419 TIANgel Mini DNA Purification Kit Kit Contents (Spin column) Cat. no. DP208 Contents
More informationGenomic Sequencing. Genomic Sequencing. Maj Gen (R) Suhaib Ahmed, HI (M)
Maj Gen (R) Suhaib Ahmed, HI (M) The process of determining the sequence of an unknown DNA is called sequencing. There are many approaches for DNA sequencing. In the last couple of decades automated Sanger
More informationPractical Of Genetics
Practical Of Genetics To learn how to extract plasmid DNA from E.coli and to observe the analysis of plasmid DNA by gel electrophoresis. Over the past decades it became evident that virtually in all bacterial
More informationLab 5/5a Transformation of E. coli with a Recombinant Plasmid
Lab 5/5a Transformation of E. coli with a Recombinant Plasmid Lab 2 Pre Lab Readiness Familiarity and Proper use of micropipettes Remember the 1 st and 2 nd stops Aseptic Technique Antibiotic Resistance
More informationBIOTECHNOLOGY. Sticky & blunt ends. Restriction endonucleases. Gene cloning an overview. DNA isolation & restriction
BIOTECHNOLOGY RECOMBINANT DNA TECHNOLOGY Recombinant DNA technology involves sticking together bits of DNA from different sources. Made possible because DNA & the genetic code are universal. 2004 Biology
More informationIntroduction. Kit components. 50 Preps GF-PC-050. Product Catalog No. 200 Preps GF-PC Preps GF-PC Preps SAMPLE
Introduction The GF-1 PCR Clean Up Kit is a system designed for rapid clean up of DNA bands ranging from 100bp to 20kb. The GF-1 PCR Clean Up Kit contains special buffers to provide the correct salt concentration
More informationThe Production of a Recombinant Biotechnology Product. Chapter 8
The Production of a Recombinant Biotechnology Product Chapter 8 Objectives Give a basic overview of genetic engineering. Describe the processes involved in isolating a piece DNA of interest Mass producing
More informationNon-Organic-Based Isolation of Mammalian microrna using Norgen s microrna Purification Kit
Application Note 13 RNA Sample Preparation Non-Organic-Based Isolation of Mammalian microrna using Norgen s microrna Purification Kit B. Lam, PhD 1, P. Roberts, MSc 1 Y. Haj-Ahmad, M.Sc., Ph.D 1,2 1 Norgen
More informationPolymerase Chain Reaction
Polymerase Chain Reaction Amplify your insert or verify its presence 3H Taq platinum PCR mix primers Ultrapure Water PCR tubes PCR machine A. Insert amplification For insert amplification, use the Taq
More informationFigure 1. Map of cloning vector pgem T-Easy (bacterial plasmid DNA)
Texas A&M University-Corpus Christi CHEM4402 Biochemistry II Laboratory Laboratory 6: Ligation & Bacterial Transformation (Bring your text and laptop to class if you wish to work on your assignment during
More informationUBC13 Cloning from Tetrahymena Thermophila. Jessie Meyer. Fall 2009
UBC13 Cloning from Tetrahymena Thermophila Jessie Meyer Fall 2009 Abstract There have been times that DNA damage has occurred during cell replication. However, To counter this threat and maintain genome
More informationRAD51 in Tetrahymena thermophila Allison Dwyre and Jenna Culver Fall 2011
Abstract RAD51 in Tetrahymena thermophila Allison Dwyre and Jenna Culver Fall 2011 From other experiments on RAD51 it can be deduced that the gene, in most mammals, is responsible for the repair of broken
More informationPCR-ReIated Products User's Instruction
ISO 9001:2000 Certified PCR-ReIated Products User's Instruction SBS Genetech Co.,Ltd. Table of Contents Cat. No. Product Name Page EUT-500 ER-500 EP-500 EQ2.2-100/2.5-100/5.2-100/5.5-100 EN-1/2 U-Taq DNA
More informationProtocol CRISPR Genome Editing In Cell Lines
Protocol CRISPR Genome Editing In Cell Lines Protocol 2: HDR donor plasmid applications (gene knockout, gene mutagenesis, gene tagging, Safe Harbor ORF knock-in) Notes: 1. sgrna validation: GeneCopoeia
More informationSTANDARD CLONING PROCEDURES. Shotgun cloning (using a plasmid vector and E coli as a host).
STANDARD CLONING PROCEDURES Shotgun cloning (using a plasmid vector and E coli as a host). 1) Digest donor DNA and plasmid DNA with the same restriction endonuclease 2) Mix the fragments together and treat
More informationTaKaRa MiniBEST Plasmid Purification Kit Ver.4.0
Cat. # 9760 For Research Use TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0 Product Manual Table of Contents I. Description... 3 II. Kit Components... 3 III. Shipping and Storage... 4 IV. Preparation
More informationPCR Detection of Genetically Modified (GM) Foods Protocol
PCR Detection of Genetically Modified (GM) Foods Protocol Purpose Isolate DNA from corn-based food so that the Polymerase Chain Reaction can be used to determine whether the selected foods have been genetically
More informationBootcamp: Molecular Biology Techniques and Interpretation
Bootcamp: Molecular Biology Techniques and Interpretation Bi8 Winter 2016 Today s outline Detecting and quantifying nucleic acids and proteins: Basic nucleic acid properties Hybridization PCR and Designing
More informationE.Z.N.A. Yeast Plasmid Mini Kit. D preps D preps
E.Z.N.A. Yeast Plasmid Mini Kit D3376-00 5 preps D3376-01 50 preps November 2015 E.Z.N.A. Yeast Plasmid Mini Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Preparing
More informationMOLECULAR BIOLOGY GREATEST HITS. Marketplace. Essentials Tour molecular biology. thermofisher.com/marketplace
molecular biology Marketplace MOLECULAR BIOLOGY GREATEST HITS Special offers on Thermo Scientific products: Cloning kits Restriction and modifying enzymes IPTG and X-Gal DNA ladders Agarose tablets Nucleic
More informationSession 7 Glycerol Stocks & Sequencing Clones
Session 7 Glycerol Stocks & Sequencing Clones Learning Objective: In this lab you will prepare several of your clones for DNA sequencing and make glycerol stock cultures as a stable and uniform starting
More informationBio 121 LAB 11 INSTRUCTIONS - DNA II
Bio 121 LAB 11 INSTRUCTIONS - DNA II In the first part of today's lab we will demonstrate that the DNA which we extracted last week can create heritable changes in the phenotype of bacterial cells. We
More informationGel Extraction Mini Spin Column Kit. UltraPrep Gel-Ex. Purification of DNA fragments and plasmids from agarose gels
Gel Extraction Mini Spin Column Kit UltraPrep Gel-Ex Purification of DNA fragments and plasmids from agarose gels 2006 Molzym, all rights reserved 1 UltraPrep Gel-Ex Manual 01/2006 Contents Kit contents
More informationEZ-10 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK
EZ-0 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK (Bacteria, Plant, Animal, Blood) Version 5.0 Rev 03/25/205 Table of Contents Introduction 2 Limitations of Use 2 Features 2 Applications 2 Storage 2
More informationLecture Four. Molecular Approaches I: Nucleic Acids
Lecture Four. Molecular Approaches I: Nucleic Acids I. Recombinant DNA and Gene Cloning Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is incorporated into a single
More informationHigh Pure Technology and Silica Adsorption High Pure PCR Product Purification Kit
for purification of DNA from PCR reactions Cat. No. 1 73 668 (50 purifications) Cat. No. 1 73 676 (50 purifications) Principle In the presence of chaotropic salt, product DNA binds selectively to glass
More informationGENERAL BIOLOGY LABORATORY II
Weeks 9-10: Bioassays of major biomolecules: Nucleic acids GENERAL BIOLOGY LABORATORY II Canbolat Gürses, Hongling Yuan, Samet Kocabay, Hikmet Geckil Department of Molecular Biology and Genetics Inonu
More informationRapid amplification of cdna ends (RACE)
Rapid amplification of cdna ends (RACE) Rapid amplification of cdna ends (RACE) is a technique used in molecular biology to obtain the full length sequence of an RNA transcript found within a cell. RACE
More informationModifications to EXPERIMENTS 21 and 24: PCR and Molecular Cloning
Modifications to EXPERIMENTS 21 and 24: PCR and Molecular Cloning This experiment was designed by Dylan Dodd, based on research completed in Dr. Isaac Cann s lab*, with modifications and editing of content
More informationChapter 20 Recombinant DNA Technology. Copyright 2009 Pearson Education, Inc.
Chapter 20 Recombinant DNA Technology Copyright 2009 Pearson Education, Inc. 20.1 Recombinant DNA Technology Began with Two Key Tools: Restriction Enzymes and DNA Cloning Vectors Recombinant DNA refers
More informationI-Blue Midi Plasmid Kit. I-Blue Midi Plasmid Kit. (Endotoxin Free) IBI SCIENTIFIC. Instruction Manual Ver For Research Use Only.
Instruction Manual Ver. 05.11.17 For Research Use Only I-Blue Midi Plasmid Kit & I-Blue Midi Plasmid Kit (Endotoxin Free) IB47180, IB47190 (2 Preparation Sample Kit) IB47181, IB47191 (25 Preparation Kit)
More informationCyber DNA Extraction and Gel Electrophoresis
Cyber DNA Extraction and Gel Electrophoresis Contributors Adam Fleming Graduate Student Georgia Southern University, GA Cheri Alderman Partner Teacher Effingham High School, GA Intended Audience K-4 5-8
More informationFROM EXPERIMENTS IN BACTERIAL GENETICS AND GENE TECHNIQUE
Uppsala 2001-04-01 REPORT FROM EXPERIMENTS IN BACTERIAL GENETICS AND GENE TECHNIQUE Laboratory assistants: Maria Jönsson Amera Gibreel Students: Contents ASSIGNMENT:... 3 INTRODUCTION:... 3 MATERIAL AND
More informationPresto Soil DNA Extraction Kit
Instruction Manual Ver. 02.23.17 For Research Use Only Presto Soil DNA Extraction Kit Advantages SLD004 (4 Preparation Sample Kit) SLD050 (50 Preparation Kit) SLD100 (100 Preparation Kit) Sample: 250-500
More informationml recombinant E. coli cultures (at a density of A 600 units per ml)
for purification of plasmid DNA, from bacterial cultures Cat. No. 1 754 777 (50 purifications) Cat. No. 1 754 785 (50 purifications) Principle Alkaline lysis releases plasmid DNA from bacteria and RNase
More informationHigh Pure RNA Isolation Kit for isolation of total RNA from 50 samples Cat. No
for isolation of total RNA from 50 samples Cat. No. 1 88 665 Principle A single reagent lyses the sample lysis and inactivates RNase. In the presence of a chaotropic salt (guanidine HCl), the released
More informationE.Z.N.A. M13 DNA Mini Kit
E.Z.N.A. M13 DNA Mini Kit D6900-00 5 preps D6900-01 50 preps EZ-96 M13 DNA Kit D1900-00 2 preps D1900-01 6 preps June 2013 E.Z.N.A. M13 DNA Mini Kit E-Z 96 M13 DNA Kit Table of Contents Introduction and
More informationFast and reliable purification of up to 100 µg of transfection-grade plasmid DNA using a spin-column.
INSTRUCTION MANUAL ZymoPURE Plasmid Miniprep Kit Catalog Nos. D4209, D4210, D4211 & D4212 (Patent Pending) Highlights Fast and reliable purification of up to 100 µg of transfection-grade plasmid DNA using
More informationAgarose Gel Electrophoresis of DNA. By: Sahar alsubaie
Agarose Gel Electrophoresis of DNA By: Sahar alsubaie principle : Agarose Gel Electrophoresis uses electrical field to separate macromolecules (DNA and Protein) that differ in size, charge and configuration.
More informationMolecular Methods in Microbial Ecology
Molecular Methods in Microbial Ecology Contact Info: Julie Huber Lillie 305 x7291 jhuber@mbl.edu Schedule: 26 Oct: Introductory Lecture, DNA extraction 28 Oct: Run DNA products on gel Lecture on PCR Prepare
More informationFigure 1: E. Coli lysate transfer using liquid handling automation
Figure 1: E. Coli lysate transfer using liquid handling automation Figure 1 - E. coli lysate transfer using liquid handling automation. Following the manufacturer s procedures, a 96-well plate miniprep
More informationGeneaid Maxi Plasmid Kit & Geneaid Maxi Plasmid Kit (Endotoxin Free)
Geneaid Maxi Plasmid Kit & Geneaid Maxi Plasmid Kit (Endotoxin Free) PM002, PME02 (2 Preparation Sample Kit) PM010, PME10 (10 Preparation Kit) PM025, PME25 (25 Preparation Kit) Instruction Manual Ver.
More informationHiPer Transformation Teaching Kit
HiPer Transformation Teaching Kit Product Code: HTBM017 Number of experiments that can be performed: 10 Duration of Experiment: 4 days Day 1- Preparation of media and revival of E. coli Host Day 2- Inoculation
More informationProblem Set 8. Answer Key
MCB 102 University of California, Berkeley August 11, 2009 Isabelle Philipp Online Document Problem Set 8 Answer Key 1. The Genetic Code (a) Are all amino acids encoded by the same number of codons? no
More informationqpcr Kit, DNA-free Product components 100 rxn 250 rxn Product description
qpcr Kit, DNA-free For the PCR detection and identification of bacterial and fungal DNA using custom primers Product code A8514 Product components 100 rxn 250 rxn A 2.5x mastermix (3 mm MgCl 2 final concentration)
More informationTable of contents. I. Description...2. Kit Components...2. Storage...2. Required reagents and equipment...2. V. Protocol...3. Example Experiment...
PCR FLT3/ITD Mutation Detection Set Cat.# 6632 Table of contents I. Description...2 II. III. IV. Kit Components...2 Storage...2 Required reagents and equipment...2 V. Protocol...3 VI. XII. Example Experiment...4
More informationChapter 10 (Part II) Gene Isolation and Manipulation
Biology 234 J. G. Doheny Chapter 10 (Part II) Gene Isolation and Manipulation Practice Questions: Answer the following questions with one or two sentences. 1. What does PCR stand for? 2. What does the
More informationTOMTEC. QUADRA 3 February 12, Application Note
QUADRA 3 February 12, 2004 Application Note Automation of Filtration Protocols The application flexibility of the Quadra 3 liquid handling workstation is enhanced with multiple application specific accessories.
More informationOverview of Current Molecular Biology Techniques
Overview of Current Molecular Biology Techniques VRSP Journal Club June 5, 2017 Chester McDowell Outline DNA preparation and analysis RNA preparation and analysis RNA-Protein Complexes Proteins Cell Culture
More informationPlasmid DNA Extraction Miniprep Kit
BIO BASIC Plasmid DNA Extraction Miniprep Kit 9K-006-0009s (10 preps) 9K-006-0009 (100 preps) 9K-006-0010 (200 preps) For Research Use Only Index Plasmid DNA Extraction Miniprep Kit Introduction 3 Important
More informationMultiple choice questions (numbers in brackets indicate the number of correct answers)
1 Multiple choice questions (numbers in brackets indicate the number of correct answers) February 1, 2013 1. Ribose is found in Nucleic acids Proteins Lipids RNA DNA (2) 2. Most RNA in cells is transfer
More informationPlantDirect TM Multiplex PCR System
PlantDirect TM Multiplex PCR System Technical Manual No. 0178 Version 10112010 I Description.. 1 II Applications 2 III Key Features.. 3 IV Shipping and Storage. 3 V Simplified Procedures. 3 VI Detailed
More informationDesigning and creating your gene knockout Background The rada gene was identified as a gene, that when mutated, caused cells to become hypersensitive
Designing and creating your gene knockout Background The rada gene was identified as a gene, that when mutated, caused cells to become hypersensitive to ionizing radiation. However, why these mutants are
More informationE.Z.N.A. HP Plasmid DNA Mini Kit I
E.Z.N.A. HP Plasmid DNA Mini Kit I D7043-00 5 preps V - Spin D7043-01 50 preps V - Spin E.Z.N.A. HP Plasmid DNA Mini Kit II D7045-00 5 preps V - Spin D7045-02 200 preps V - Spin July 2013 E.Z.N.A. HP
More informationRapid and Efficient RNA Isolation Protocol from Various Recalcitrant Tissues of Mango (Mangifera indica L.)
Rapid and Efficient RNA Isolation Protocol from Various Recalcitrant Tissues of Mango (Mangifera indica L.) R.R. Sharma and S. V. R. Reddy Division of Food Science and Postharvest Technology, ICAR-Indian
More informationCloning Tetrahymena Gene THD9 Bridget Tabora & Caitlin Roam Fall 2009
Cloning Tetrahymena Gene THD9 Bridget Tabora & Caitlin Roam Fall 2009 Abstract: HDACs are enzymes that modify histones and regulate gene expression. Sirtuins are a specific class of HDAC, and they relate
More informationPrepare CTAB solutions to extracting DNA from Plant
Prepare CTAB solutions to extracting DNA from Plant By Dr. Mona S. Alwahibi Botany and Microbiology Dep. Introduction The search for a more efficient means of extracting DNA of both higher quality and
More informationConstruction of a Mutant pbr322 Using Site-Directed Mutagenesis to Investigate the Exclusion Effects of pbr322 During Co-transformation with puc19
Construction of a Mutant pbr322 Using Site-Directed Mutagenesis to Investigate the Exclusion Effects of pbr322 During Co-transformation with puc19 IVANA KOMLJENOVIC Department of Microbiology and Immunology,
More informationMOLECULAR BIOLOGY EXPERIMENT PCR & SEEDING
BME MOLECULAR BIOLOGY EXPERIMENT PCR & SEEDING SKKU BME 3 RD GRADE, 2 ND SEMESTER FOR FUN PCR & SEEDING PCR: polymerase chain reaction Electrophoresis Seeding These are for amplifying DNA and cell PCR
More informationIsolation of genomic DNA from buccal swabs - a brief protocol. Assessment of DNA concentration and purity
Molecular biology 1 DNA Isolation Isolation of genomic DNA from buccal swabs - a brief protocol MACHEREY-NAGEL isolation kit Protocol: 1. Gently rub and rotate swab along the inside of the cheek (both
More informationPuro. Knockout Detection (KOD) Kit
Puro Knockout Detection (KOD) Kit Cat. No. CC-03 18 Oct. 2016 Contents I. Kit Contents and Storage II. Product Overview III. Methods Experimental Outline Genomic DNA Preparation Obtain Hybrid DNA Digest
More informationApplication Note 18 RNA/DNA/Protein Sample Preparation METHODS AND MATERIALS INTRODUCTION
Application Note 18 /DNA/Protein Sample Preparation Sequential Purification of, DNA and Protein from a Single Sample using 's /DNA/Protein Purification Kit and Comparison to a Market B. Lam, PhD 1, C.
More informationDevelopment of Positive Control for Hepatitis B Virus
Human Journals Research Article December 2015 Vol.:2, Issue:2 All rights are reserved by Saurabh Bandhavkar et al. Development of Positive Control for Hepatitis B Virus Keywords: Hepatitis B virus, pbluescript,
More informationImpact of Nutraceuticals on TERT gene encoded protein
Impact of Nutraceuticals on TERT gene encoded protein Xu Liu Department of Biological Sciences Fordham University, Bronx, New York, 10458 Abstract Telomerase is a Ribonucleo-protein polymerase that plays
More informationComputational Biology I LSM5191
Computational Biology I LSM5191 Lecture 5 Notes: Genetic manipulation & Molecular Biology techniques Broad Overview of: Enzymatic tools in Molecular Biology Gel electrophoresis Restriction mapping DNA
More informationPlasmid Maxiprep Plus Purification Kit
Plasmid Maxiprep Plus Purification Kit Cat. # : DP01MX-P10/ DP01MX-P20 Size : 10/20 Reactions Store at RT For research use only 1 Description: The Plasmid Maxiprep Plus Purification Kit provides simple,
More information