Electrophoresis, cleaning up on spin-columns, labeling of PCR products and preparation extended products for sequencing
|
|
- Briana Floyd
- 7 years ago
- Views:
Transcription
1 Electrophoresis, cleaning up on spin-columns, labeling of PCR products and preparation extended products for sequencing PAGE electrophoresis Polyacrylamide gel electrophoresis (PAGE) is used for separating molecules by size (DNA or proteins). Pore size is determined by the ratio of acrylamide and bis-acrylamide concentra-tion. Care must be taken when working with acrylamide, since it is a strong neurotoxin, espe-cially in its powdered form. Polyacrylamide gels can separate DNA that differs by 0.2% in length, well beyond the resolving capabilities of agarose (2%). Another advantage of using polyacrylamide gels is that they can accommodate large amounts of DNA (up to 10 μg) without any loss in resolution. Depending upon the application, TBE gels can be prepared as denaturing or nondenaturing ones. Purification of amplicons Purification of nucleic acids using a specially selected silica membrane is a rapid, conven-ient and economical way to isolate DNA and RNA. The technique does not require time-consuming methods based on old technologies such as: use of extraction methods in the phe-nol-chloroform system, precipitation, or PEG-type polymers. Applied membrane technology allows to avoid problems with the silica particles used often as suspensions. The purification procedure removes primers, nucleotides, enzymes, mineral oils, salts, agarose, ethidium bro-mide, and other impurities from DNA samples. The Clean-Up kit is based on the DNA ability to adsorb to silica surface in the presence of high concentration of chaotropic salts [Fig.1]. Starting up the DNA recovery after enzymatic reaction, the binding solution G which contains chaotropic salt, is added to the reaction mix-ture. At high salt concentrations, sodium cations break hydrogen bonds between the hydro-gen atoms of water and the negatively charged oxygen ions in silica. Sodium serves as a cati-on bridge and interacts with negatively charged oxygen of the phosphate backbone of the DNA molecule.
2 During the next step the whole sample is loaded onto minicolumn with dedicated silica res-in. The DNA binds to the silica resin, while contaminants (enzymes, salts, short primers,etc.) are passing through the column freely. Leftovers of contaminants are moved away in the washing step. Pure DNA is eluted at low salt buffer or water and can be used in the following experiments without additional purification procedures (e.g. precipitation). DNA Chain Termination Sequencing The chain-terminator method (sometimes referred to as the Sanger method) is more effi-cient and uses fewer toxic chemicals and lower amounts of radioactivity than the method of Maxam and Gilbert. Generally it is method of choice for DNA sequencing. The key principle of the Sanger method was the use of dideoxynucleotides triphosphates (ddntps) as DNA chain termination [Figs 3 and 4]. The classical chain-termination method [Fig. 2.] requires a single-stranded DNA template, a DNA primer, a DNA polymerase, radioactively or fluorescently labeled nucleotides and modified nucleotides that terminate DNA strand elongation. The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (datp, dgtp, dctp and dttp) and the DNA polymerase. To each reaction added is only one of the four dideoxynucleotides (ddatp, ddgtp, ddctp, or ddttp) which are the chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, thus terminating DNA strand extension and providing various DNA fragments of varying length. Dye-terminator sequencing utilizes labelling of the chain terminator ddntps, which per-mits sequencing in a single reaction, rather than four reactions as in the labelled-primer meth-od [Fig. 3]. Dye-terminator sequencing requires each of the four dideoxynucleotide chain terminators to be labelled with different fluorescent dyes (different wavelengths of fluores-cence emission) [Fig. 4]. Owing to its greater expediency and speed, dye-terminator sequenc-ing is now the mainstay in automated sequencing. Its limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromato-gram after capillary electrophoresis. The dye-terminator sequencing method, along with au-tomated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects.
3
4 Purification of chain terminated fragments The best results are obtained when unincorporated dye terminators are completely removed prior to capillary electrophoresis. Excess dye terminators, primers, proteins in sequencing reactions obscure data in the early part of the sequence and can interfere with basecalling. When unincorporated dye is present in large amount then on sequence will be visible dye blobs. Fig. 4. Example of electropherogram with visible dye blobs This are the most popular methods used for purification: Ethanol/EDTA precipitation Ethanol/EDTA/sodium acetate precipitation Plate or spin column purification Used in this training method is based on precipitation of cycle sequencing reaction products on the surface of especially designed membrane. The addition of Mix Blue reagent to the cycle sequencing reaction mixture enables an easy control of precipitation process. In the next step the binding-washing solution is added and the whole mixture is loaded onto Exterminator spin column. During the short centrifugation step the cycle sequencing products are bound by the spin column membrane while the excess of non-incorporated nucleotide terminators and sequencing primer pass through the membrane. The remaining salts and leftovers of other impurities are removed in the washing step. Subsequently the purified cycle sequencing reaction products are eluted directly by water. Literatura: Brody, J.R., Kern, S.E. (2004) History and principles of conductive media for standard DNA electrophoresis. Anal Biochem. 333(1):1-13 Smith LM, Sanders JZ, Kaiser RJ, et al (1986). "Fluorescence detection in automated DNA sequence analysis". Nature 321 (6071): Maxam AM, Gilbert W (1977). "A new method for sequencing DNA". Proc. Natl. Acad. Sci. U.S.A. 74 (2): Carter, J.M. and Milton, I.D., (1993) "An inexpensive and simple method for DNA purifications on silica particles", Nucl. Acids Res.21
5 MATERIALS REAGENTS - EQUIPMENT thermal cycler thermoblock centrifuge vortex primers NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) Ex-Terminator (A&A Biotechnology) BigDye Terminator v3.1 Cycle Sequencing Kit 1,5 ml tubes 10x TBE electrophoresis buffer (0,1 M Tris, 83 mm Boric acid, 1 mm EDTA) plates with 10% polyacrylamide gel gelloading buffer (0,25 % Bromophenol blue, 0,25 % Xylene cyanol and 40 % sucrose) vertical electrophoresis apparatus (Bio-Rad Mini Protean II) reagent A (EtOH, CH3COOH) reagent B (AgNO3-0,17 %) reagent C (NaOH - 10%, Formaldehyde CH2O - 2%) PROCEDURE PAGE Electrophoresis 1. Clamp your prepared gels and fill up buffer chambers with electrophoresis buffer 1x TBE. 2. Mix the DNA samples (3,5 µl) with the appropriate amount of gelloading buffer (1µl). 3. Load the mixture into the wells using a micropipette. (It is important not to take too long to complete loading the gel; otherwise, the samples will diffuse from the wells) 4. Connect the electrodes to a power pack, turn on the power, and begin the electrophoresis run (Adjust electric current to 30 ma/gel plate) 5. Run the gel until the marker dyes have migrated the desired distance. Turn off the electric power, disconnect the leads, and discard the electrophoresis buffer from the reservoirs. 6. Detach the glass plates. Lay the glass plates on the bench. Use a spacer or plastic wedge to lift a corner of the glass plate. 7. Put gels into the boxes filled with water. Silver staining 1. Wash the gel in H2O for at least 5 min. 2. Discard water and fill up the box with reagent A for 5 min. 3. Discard solution A and fill up the gel with reagent B for 10min. 4. Next rinse the gel with water 3x for 1 minute. 5. Fill up gel with reagent C and instantly discard it. Repeat this procedure until solution is no longer black. 6. Leave the gel in transparent reagent C until the fragments of DNA are visible. 7. Next fill up the gel with water and interpret results.
6 Clean up amplicons on spin columns
7 Use the obtained cleaned fragments to prepare reaction with BigDye. DNA Chain Termination Sequencing Reaction 1. Combine the following component for each reaction in a 0.2 ml tube (add BigDye at the end): Reagent Volume Concentration H 2O 3,75 µl - Sequencing buffer 3,5 µl 5x Primer 0,25 µl 10 µm BigDye Terminator v3.1 Ready Reaction Mix 0,5 µl - Clean PCR produkt 2 µl - 2. Use following primers to specific reaction: (EXI, Left primer), (EXII, Right primer), (Mitochondrial long HVR fragment, Left Primer) 3. Place tubes in a thermal cycler preheated to 95 C and start with proper times and temperature as stated below:
8 HVR Number of cycle 35 Initial denaturation 95ºC 5min Denaturation 95ºC 30 sec Anneling 58ºC 7 sec Extention 60ºC 4 min Hold 4ºC Purifying of extended products (ExTerminator kit) 1. Add 5 μl of Mix Blue to cycle sequencing reaction mixture. 2. Add 100 μl of bind/wash solution and mix sample by pipetting and load the whole sample onto minicolumn. 3. Spin at 1500 x g for 30 s. (the light blue colour of minicolumn membrane is a result of efficient precipitation of sequencing products.) 4. Apply on minicolumn 400 μl of bind/wash solution. 5. Spin at 6000 x g for 2 min. 6. Transfer the minicolumn to a new 1.5 ml tube 7. Apply precisely onto the membrane (center of dark blue circle) 15 μl of water. 8. Incubate at RT for 2 min. 9. Spin at 6000 x g for 2 min. (Light blue colour of eluted sample confirms proper purification of sample) 10. Dry the sample in thermoblock 55 C. 11. Dissolve dried pallet in 12µl of deionized formamide.
PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System) 50 preps Product #60200
3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System)
More information1/12 Dideoxy DNA Sequencing
1/12 Dideoxy DNA Sequencing Dideoxy DNA sequencing utilizes two steps: PCR (polymerase chain reaction) amplification of DNA using dideoxy nucleoside triphosphates (Figures 1 and 2)and denaturing polyacrylamide
More informationZR DNA Sequencing Clean-up Kit
INSTRUCTION MANUAL ZR DNA Sequencing Clean-up Kit Catalog Nos. D40 & D4051 Highlights Simple 2 Minute Bind, Wash, Elute Procedure Flexible 6-20 µl Elution Volumes Allow for Direct Loading of Samples with
More informationZR-96 DNA Sequencing Clean-up Kit Catalog Nos. D4052 & D4053
INSTRUCTION MANUAL ZR-96 DNA Sequencing Clean-up Kit Catalog Nos. D4052 & D4053 Highlights Simple 10 Minute Bind, Wash, Elute Procedure Flexible 15-20 µl Elution Volumes Allow for Direct Loading of Samples
More informationISOLATE II PCR and Gel Kit. Product Manual
ISOLATE II PCR and Gel Kit Product Manual 2 Product Manual www.bioline.com/isolate PCR and Gel Kit ISOLATE II PCR and Gel Kit ISOLATE II PCR and Gel Kit 1 Kit contents 04 2 Description 04 3 Storage 04
More information- In 1976 1977, Allan Maxam and walter Gilbert devised the first method for sequencing DNA fragments containing up to ~ 500 nucleotides.
DNA Sequencing - DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases adenine, guanine, cytosine, and thymine in a molecule of DNA. -
More informationDNA SEQUENCING (using an ABI automated sequencer)
DNA SEQUENCING (using an ABI automated sequencer) OBTECTIVE: To label and separate DNA fragments varying by single nucleotides, in order to determine the sequence of nucleotides. INTRODUCTION: Determination
More informationTIANquick Mini Purification Kit
TIANquick Mini Purification Kit For purification of PCR products, 100 bp to 20 kb www.tiangen.com TIANquick Mini Purification Kit (Spin column) Cat no. DP203 Kit Contents Contents Buffer BL Buffer PB Buffer
More informationThe Techniques of Molecular Biology: Forensic DNA Fingerprinting
Revised Fall 2011 The Techniques of Molecular Biology: Forensic DNA Fingerprinting The techniques of molecular biology are used to manipulate the structure and function of molecules such as DNA and proteins
More informationSanger Sequencing and Quality Assurance. Zbigniew Rudzki Department of Pathology University of Melbourne
Sanger Sequencing and Quality Assurance Zbigniew Rudzki Department of Pathology University of Melbourne Sanger DNA sequencing The era of DNA sequencing essentially started with the publication of the enzymatic
More informationSanger Sequencing: Sample Preparation Guide
Sanger Sequencing: Sample Preparation Guide Use this as a guide to prepare your samples for Sanger sequencing at AGRF CONTENTS 1 Overview... 2 1.1 Capillary Separation (CS) or electrophoretic separation
More informationWizard DNA Clean-Up System INSTRUCTIONS FOR USE OF PRODUCT A7280.
Technical Bulletin Wizard DNA Clean-Up System INSTRUCTIONS FOR USE OF PRODUCT A7280. PRINTED IN USA. Revised 4/06 AF9TB141 0406TB141 Wizard DNA Clean-Up System All technical literature is available on
More informationTroubleshooting Sequencing Data
Troubleshooting Sequencing Data Troubleshooting Sequencing Data No recognizable sequence (see page 7-10) Insufficient Quantitate the DNA. Increase the amount of DNA in the sequencing reactions. See page
More informationID kit. imegen Anchovies II. and E. japonicus) DNA detection by. User manual. Anchovies species (E. encrasicolus. sequencing.
User manual imegen Anchovies II ID kit Anchovies species (E. encrasicolus and E. japonicus) DNA detection by sequencing Reference: Made in Spain The information in this guide is subject to change without
More informationSouthern Blot Analysis (from Baker lab, university of Florida)
Southern Blot Analysis (from Baker lab, university of Florida) DNA Prep Prepare DNA via your favorite method. You may find a protocol under Mini Yeast Genomic Prep. Restriction Digest 1.Digest DNA with
More informationTroubleshooting Guide for DNA Electrophoresis
Troubleshooting Guide for Electrophoresis. ELECTROPHORESIS Protocols and Recommendations for Electrophoresis electrophoresis problem 1 Low intensity of all or some bands 2 Smeared bands 3 Atypical banding
More informationDNA Separation Methods. Chapter 12
DNA Separation Methods Chapter 12 DNA molecules After PCR reaction produces many copies of DNA molecules Need a way to separate the DNA molecules from similar sized molecules Only way to genotype samples
More informationMinElute Handbook. Sample & Assay Technologies. March 2008
March 2008 MinElute Handbook MinElute PCR Purification Kit For purification of PCR products (70 bp to 4 kb) in low elution volumes MinElute Gel Extraction Kit For gel extraction of DNA fragments (70 bp
More informationObjectives: Vocabulary:
Introduction to Agarose Gel Electrophoresis: A Precursor to Cornell Institute for Biology Teacher s lab Author: Jennifer Weiser and Laura Austen Date Created: 2010 Subject: Molecular Biology and Genetics
More informationGenomic DNA Clean & Concentrator Catalog Nos. D4010 & D4011
Page 0 INSTRUCTION MANUAL Catalog Nos. D4010 & D4011 Highlights Quick (5 minute) spin column recovery of large-sized DNA (e.g., genomic, mitochondrial, plasmid (BAC/PAC), viral, phage, (wga)dna, etc.)
More informationThe Biotechnology Education Company
EDVTEK P.. Box 1232 West Bethesda, MD 20827-1232 The Biotechnology 106 EDV-Kit # Principles of DNA Sequencing Experiment bjective: The objective of this experiment is to develop an understanding of DNA
More informationIntroduction. Preparation of Template DNA
Procedures and Recommendations for DNA Sequencing at the Plant-Microbe Genomics Facility Ohio State University Biological Sciences Building Room 420, 484 W. 12th Ave., Columbus OH 43210 Telephone: 614/247-6204;
More informationUltraClean Soil DNA Isolation Kit
PAGE 1 UltraClean Soil DNA Isolation Kit Catalog # 12800-50 50 preps New improved PCR inhibitor removal solution (IRS) included Instruction Manual (New Alternative Protocol maximizes yields) Introduction
More informationPlant Genomic DNA Extraction using CTAB
Plant Genomic DNA Extraction using CTAB Introduction The search for a more efficient means of extracting DNA of both higher quality and yield has lead to the development of a variety of protocols, however
More informationSequencing Guidelines Adapted from ABI BigDye Terminator v3.1 Cycle Sequencing Kit and Roswell Park Cancer Institute Core Laboratory website
Biomolecular Core Facility AI Dupont Hospital for Children, Rockland Center One, Room 214 Core: (302) 651-6712, Office: (302) 651-6707, mbcore@nemours.org Katia Sol-Church, Ph.D., Director Jennifer Frenck
More informationEZ Load Molecular Rulers. Catalog Numbers 170-8351 20 bp 170-8352 100 bp 170-8353 100 bp PCR 170-8354 500 bp 170-8355 1 kb 170-8356 Precision Mass
EZ Load Molecular Rulers Catalog Numbers 170-8351 20 bp 170-8352 100 bp 170-8353 100 bp PCR 170-8354 500 bp 170-8355 1 kb 170-8356 Precision Mass EZ Load Molecular Rulers Quantity DNA sufficient for 100
More informationLab 5: DNA Fingerprinting
Lab 5: DNA Fingerprinting You are about to perform a procedure known as DNA fingerprinting. The data obtained may allow you to determine if the samples of DNA that you will be provided with are from the
More informationPyroPhage 3173 DNA Polymerase, Exonuclease Minus (Exo-)
PyroPhage 3173 DNA Polymerase, Exonuclease Minus (Exo-) FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE Lucigen Corporation 2905 Parmenter St, Middleton, WI 53562 USA Toll Free: (888) 575-9695 (608)
More information50 g 650 L. *Average yields will vary depending upon a number of factors including type of phage, growth conditions used and developmental stage.
3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com Phage DNA Isolation Kit Product # 46800, 46850 Product Insert
More informationHiPer 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 informationIn vitro analysis of pri-mirna processing. by Drosha-DGCR8 complex. (Narry Kim s lab)
In vitro analysis of pri-mirna processing by Drosha-DGCR8 complex (Narry Kim s lab) 1-1. Preparation of radiolabeled pri-mirna transcript The RNA substrate for a cropping reaction can be prepared by in
More informationDNA: A Person s Ultimate Fingerprint
A partnership between the UAB Center for Community Outreach Development and McWane Center DNA: A Person s Ultimate Fingerprint This project is supported by a Science Education Partnership Award (SEPA)
More informationGENOTYPING ASSAYS AT ZIRC
GENOTYPING ASSAYS AT ZIRC A. READ THIS FIRST - DISCLAIMER Dear ZIRC user, We now provide detailed genotyping protocols for a number of zebrafish lines distributed by ZIRC. These protocols were developed
More informationWizard SV Gel and PCR Clean-Up System
TECHNICAL BULLETIN Wizard SV Gel and PCR Clean-Up System Instruc ons for Use of Products A9280, A9281, A9282 and A9285 Revised 12/10 TB308 Wizard SV Gel and PCR Clean-Up System All technical literature
More informationProtocol 001298v001 Page 1 of 1 AGENCOURT RNACLEAN XP IN VITRO PRODUCED RNA AND CDNA PURIFICATION
Page 1 of 1 AGENCOURT RNACLEAN XP IN VITRO PRODUCED RNA AND CDNA PURIFICATION Please refer to http://www.agencourt.com/technical for updated protocols and refer to MSDS instructions when handling or shipping
More informationUltraClean PCR Clean-Up Kit
UltraClean PCR Clean-Up Kit Catalog No. Quantity 12500-50 50 Preps 12500-100 100 Preps 12500-250 250 Preps Instruction Manual Please recycle Version: 02212013 1 Table of Contents Introduction... 3 Protocol
More informationGuide to Reverse Phase SpinColumns Chromatography for Sample Prep
Guide to Reverse Phase SpinColumns Chromatography for Sample Prep www.harvardapparatus.com Contents Introduction...2-3 Modes of Separation...4-6 Spin Column Efficiency...7-8 Fast Protein Analysis...9 Specifications...10
More informationAurora Forensic Sample Clean-up Protocol
Aurora Forensic Sample Clean-up Protocol 106-0008-BA-D 2015 Boreal Genomics, Inc. All rights reserved. All trademarks are property of their owners. http://www.borealgenomics.com support@borealgenomics.com
More informationGenolution Pharmaceuticals, Inc. Life Science and Molecular Diagnostic Products
Genolution Pharmaceuticals, Inc. Revolution through genes, And Solution through genes. Life Science and Molecular Diagnostic Products www.genolution1.com TEL; 02-3010-8670, 8672 Geno-Serum Hepatitis B
More informationHiPer Ion Exchange Chromatography Teaching Kit
HiPer Ion Exchange Chromatography Teaching Kit Product Code: HTC001 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 5-6 hours Storage Instructions: The kit is stable for
More informationProcedures For DNA Sequencing
Procedures For DNA Sequencing Plant-Microbe Genomics Facility (PMGF) Ohio State University 420 Biological Sciences Building 484 W. 12th Ave., Columbus OH 43210 Telephone: 614/247-6204 FAX: 614/292-6337
More informationMICB ABI PRISM 310 SEQUENCING GUIDE SEQUENCING OF PLASMID DNA
Plasmid DNA Preparation MICB ABI PRISM 310 SEQUENCING GUIDE SEQUENCING OF PLASMID DNA Introduction: I have always used the classic Alkaline Lysis miniprep method to isolate plasmid DNA. (See below) If
More informationAutomated High Throughput Purification of BigDye TM Terminator Fluorescent DNA Sequencing Reactions Using Wizard MagneSil TM Paramagnetic Particles
Automated High Throughput Purification of BigDye TM Terminator Fluorescent DNA Sequencing Reactions Using Wizard MagneSil TM Paramagnetic Particles Paul Otto*, Brad Larson and Steve Krueger Abstract We
More informationGenomic DNA Extraction Kit INSTRUCTION MANUAL
Genomic DNA Extraction Kit INSTRUCTION MANUAL Table of Contents Introduction 3 Kit Components 3 Storage Conditions 4 Recommended Equipment and Reagents 4 Introduction to the Protocol 4 General Overview
More informationPreparing Samples for Sequencing Genomic DNA
Preparing Samples for Sequencing Genomic DNA FOR RESEARCH ONLY Topics 3 Introduction 5 Kit Contents and Equipment Checklist 7 Fragment the Genomic DNA 11 Perform End Repair 12 Add A Bases to the 3' End
More informationab185916 Hi-Fi cdna Synthesis Kit
ab185916 Hi-Fi cdna Synthesis Kit Instructions for Use For cdna synthesis from various RNA samples This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 1
More informationFOR REFERENCE PURPOSES
BIOO LIFE SCIENCE PRODUCTS FOR REFERENCE PURPOSES This manual is for Reference Purposes Only. DO NOT use this protocol to run your assays. Periodically, optimizations and revisions are made to the kit
More informationHow To Make A Tri Reagent
TRI Reagent For processing tissues, cells cultured in monolayer or cell pellets Catalog Number T9424 Store at room temperature. TECHNICAL BULLETIN Product Description TRI Reagent is a quick and convenient
More informationRunning protein gels and detection of proteins
Running protein gels and detection of proteins 1. Protein concentration determination using the BIO RAD reagent This assay uses a colour change reaction to give a direct measurement of protein concentration.
More informationExpressArt Bacterial H-TR cdna synthesis kit. With extreme selectivity against rrnas
ExpressArt Bacterial H-TR cdna synthesis kit With extreme selectivity against rrnas suitable for 2 to 4 µg total RNA Catalogue No. 8004-A30 (30 rxns) Reagents Materials are provided for 30 cdna synthesis
More informationAxyPrep TM Mag PCR Clean-up Protocol
AxyPrep TM Mag PCR Clean-up Protocol Intro The AxyPrep Mag PCR Clean-up kit utilizes a unique paramagnetic bead technology for rapid, high-throughput purification of PCR amplicons. Using this kit, PCR
More informationLAB 11 PLASMID DNA MINIPREP
LAB 11 PLASMID DNA MINIPREP STUDENT GUIDE GOAL The objective of this lab is to perform extraction of plasmid DNA and analyze the results. OBJECTIVES After completion, the student should be able to: 1.
More informationVLLM0421c Medical Microbiology I, practical sessions. Protocol to topic J10
Topic J10+11: Molecular-biological methods + Clinical virology I (hepatitis A, B & C, HIV) To study: PCR, ELISA, your own notes from serology reactions Task J10/1: DNA isolation of the etiological agent
More informationClassic Immunoprecipitation
292PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Classic Immunoprecipitation Utilizes Protein A/G Agarose for Antibody Binding (Cat.
More informationABI PRISM BigDye Primer Cycle Sequencing Ready Reaction Kit With AmpliTaq DNA Polymerase, FS
ABI PRISM BigDye Primer Cycle Sequencing Ready Reaction Kit With AmpliTaq DNA Polymerase, FS Protocol Copyright 2003, 2010 Applied Biosystems. Printed in the U.S.A. For Research Use Only. Not for use in
More informationApplication Guide... 2
Protocol for GenomePlex Whole Genome Amplification from Formalin-Fixed Parrafin-Embedded (FFPE) tissue Application Guide... 2 I. Description... 2 II. Product Components... 2 III. Materials to be Supplied
More informationReal-time monitoring of rolling circle amplification using aggregation-induced emission: applications for biological detection
Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 215 Supplementary Information Real-time monitoring of rolling circle amplification using aggregation-induced
More informationRNA Fragment DeepSeq Library Preparation Protocol
RNA Fragment DeepSeq Library Preparation Protocol I) LIGATION Recommended input: RNA between 0.05-2 pmol; must have 3' OH 1. Thaw 10X T4 RNA Ligase Reaction Buffer, 50% PEG8000, 20 mm DTT, 7 um App Adaptor
More informationNorthern blot analysis for microrna. (Narry Kim s lab)
Northern blot analysis for microrna (Narry Kim s lab) Materials 1. 10~50 μg of total RNA extracted from HeLa cells treated with sirna 2. RNA loading buffer 3. Probe: DNA oligonucleotide complementary to
More informationDNA SPOOLING 1 ISOLATION OF DNA FROM ONION
DNA SPOOLING 1 ISOLATION OF DNA FROM ONION INTRODUCTION This laboratory protocol will demonstrate several basic steps required for isolation of chromosomal DNA from cells. To extract the chromosomal DNA,
More informationRAINBOW ELECTROPHORESIS 1 An Introduction to Gel Electrophoresis
RAINBOW ELECTROPHORESIS 1 An Introduction to Gel Electrophoresis INTRODUCTION This laboratory will demonstrate the basics of electrophoresis and the theory behind the separation of molecules on an agarose
More informationRT31-020 20 rxns. RT31-100 100 rxns TRANSCRIPTME Enzyme Mix (1) 40 µl 2 x 50 µl 5 x 40 µl
Components RT31-020 20 rxns RT31-050 50 rxns RT31-100 100 rxns TRANSCRIPTME Enzyme Mix (1) 40 µl 2 x 50 µl 5 x 40 µl 2x RT Master Mix (2) 200 µl 2 x 250 µl 5 x 200 µl RNase H (E. coli) 20 µl 2 x 25 µl
More informationFirst Strand cdna Synthesis
380PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name First Strand cdna Synthesis (Cat. # 786 812) think proteins! think G-Biosciences
More informationGenomeLab Sequencing Chemistry Protocol
TM 390003-AF July 2007 GenomeLab Sequencing Chemistry Protocol CEQ / GenomeLab Series Genetic Analysis Systems Beckman Coulter, Inc. 4300 N. Harbor Blvd., Fullerton, CA 92834-3100 Copyright 2007 Beckman
More informationSequiTherm EXCEL II DNA Sequencing Kit-LC (for 66-cm gels)
SequiTherm EXCEL II DNA Sequencing Kit-LC (for 66-cm gels) Cat. Nos. SE9202LC and SED52100 The SequiTherm EXCEL II DNA Sequencing Kit-LC (for 66-cm gels) is optimized for use with LI-COR model 4x00 and
More informationProcedure for RNA isolation from human muscle or fat
Procedure for RNA isolation from human muscle or fat Reagents, all Rnase free: 20% SDS DEPC-H2O Rnase ZAP 75% EtOH Trizol Chloroform Isopropanol 0.8M NaCitrate/1.2M NaCl TE buffer, ph 7.0 1. Homogenizer-probe
More informationDye-Blob message: Example: Generally, this is due to incomplete excess dye removal of the cycle sequence reaction.
When sequence data is uploaded to ilab, an email is sent notifying the user that data is ready. The staff of the DNA facility has the ability to edit this message to include specific remarks about how
More informationWestern Blotting: Mini-gels
Western Blotting: Mini-gels Materials a Protein Extraction Buffer (for callus or kernel), Solution Stock Final Volume Tris-HCl ph 80 1 M 200 mm 20 ml NaCl 4 M 100 mm 25 ml Sucrose 2 M 400 mm 20 ml EDTA
More informationTerra PCR Direct Polymerase Mix User Manual
Clontech Laboratories, Inc. Terra PCR Direct Polymerase Mix User Manual Cat. Nos. 639269, 639270, 639271 PT5126-1 (031416) Clontech Laboratories, Inc. A Takara Bio Company 1290 Terra Bella Avenue, Mountain
More informationDNA Sequencing. Contents. Introduction. Maxam-Gilbert
DNA Sequencing Contents Introduction... 1 Maxam-Gilbert... 1 Sanger... 3 Automated Fluorescence Sequencing... 5 References... 8 Introduction Prior to the mid-1970 s no method existed by which DNA could
More informationThe fastest spin-column based procedure for purifying up to 10 mg of ultra-pure endotoxin-free transfection-grade plasmid DNA.
INSTRUCTION MANUAL ZymoPURE Plasmid Gigaprep Kit Catalog Nos. D4204 (Patent Pending) Highlights The fastest spin-column based procedure for purifying up to 10 mg of ultra-pure endotoxin-free transfection-grade
More informationRNA Isolation for Frozen Mouse Livers and Reverse Transcription
RNA Isolation for Frozen Mouse Livers and Reverse Transcription I. Introduction RNA is typically isolated from tissue or cells based on the procedure originally described by Chomczynski and Sacchi in 1987.
More informationDNA Detection. Chapter 13
DNA Detection Chapter 13 Detecting DNA molecules Once you have your DNA separated by size Now you need to be able to visualize the DNA on the gel somehow Original techniques: Radioactive label, silver
More informationPCR Optimization. Table of Contents Fall 2012
Table of Contents Optimizing the Polymerase Chain Reaction Introduction.....1 Review of Mathematics........ 3 Solving Problems of Dilution and Concentration: Two Approaches.. 4 Experiment Overview 7 Calculations
More informationRevertAid Premium First Strand cdna Synthesis Kit
RevertAid Premium First Strand cdna Synthesis Kit #K1651, #K1652 CERTIFICATE OF ANALYSIS #K1651 Lot QUALITY CONTROL RT-PCR using 100 fg of control GAPDH RNA and GAPDH control primers generated a prominent
More informationSTRUCTURES OF NUCLEIC ACIDS
CHAPTER 2 STRUCTURES OF NUCLEIC ACIDS What is the chemical structure of a deoxyribonucleic acid (DNA) molecule? DNA is a polymer of deoxyribonucleotides. All nucleic acids consist of nucleotides as building
More informationSanger Sequencing. Troubleshooting Guide. Failed sequence
Sanger Sequencing Troubleshooting Guide Below are examples of the main problems experienced in ABI Sanger sequencing. Possible causes for failure and their solutions are listed below each example. The
More informationNimbleGen DNA Methylation Microarrays and Services
NimbleGen DNA Methylation Microarrays and Services Sample Preparation Instructions Outline This protocol describes the process for preparing samples for NimbleGen DNA Methylation microarrays using the
More informationUltraClean Forensic DNA Isolation Kit (Single Prep Format)
UltraClean Forensic DNA Isolation Kit (Single Prep Format) Catalog No. Quantity 14000-10 10 preps 14000-S 1 prep Instruction Manual Please recycle Version: 10302012 1 Table of Contents Introduction...
More informationRESTRICTION ENZYME ANALYSIS OF DNA
University of Massachusetts Medical School Regional Science Resource Center SUPPORTING MATHEMATICS, SCIENCE AND TECHNOLOGY EDUCATION 222 Maple Avenue, Stoddard Building Shrewsbury, MA 01545-2732 508.856.5097
More informationPNA BRAF Mutation Detection Kit
- PNA BRAF Mutation Detection Kit Catalog Number KA2102 50 tests/kit Version: 01 Intended for research use only www.abnova.com Introduction and Background Intended use The PNA BRAF Mutation Detection Kit
More informationSTA DARD OPERATI G PROCEDURE FOR THE DETECTIO OF AFRICA SWI E FEVER VIRUS (ASFV) BY CO VE TIO AL POLYMERASE CHAI REACTIO (PCR)
STA DARD OPERATI G PROCEDURE FOR THE DETECTIO OF AFRICA SWI E FEVER VIRUS (ASFV) BY CO VE TIO AL POLYMERASE CHAI REACTIO (PCR) jmvizcaino@vet.ucm.es Av/ Puerta de Hierro s/n. 28040 Madrid. Tel: (34) 913944082
More informationDNA Sequencing Setup and Troubleshooting
DNA Sequencing Setup and Troubleshooting Lara Cullen, PhD Scientific Applications Specialist Australia and New Zealand Reviewing Sequencing Data Review the Electropherogram Review the Raw Data (Signal
More informationBigDye Terminator v3.1 Cycle Sequencing Kit. Protocol. DRAFT August 27, 2002 12:32 pm, 4337035A_v3.1Title.fm
BigDye Terminator v3.1 Cycle Sequencing Kit Protocol August 27, 2002 12:32 pm, 4337035A_v3.1Title.fm Copyright 2002, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic
More informationMagExtractor -Genome-
Instruction manual MagExtractor-Genome-0810 F0981K MagExtractor -Genome- NPK-101 100 preparations Store at 4 C Contents [1] Introduction [2] Components [3] Materials required [4] Protocol 1. Purification
More informationDNA Sequence Analysis
DNA Sequence Analysis Two general kinds of analysis Screen for one of a set of known sequences Determine the sequence even if it is novel Screening for a known sequence usually involves an oligonucleotide
More informationHow To Get Rid Of Small Dna Fragments
AxyPrep TM Mag FragmentSelect-I Protocol (Fragment Size Selection for Illumina Genome Analyzer and Life Technologies SoLiD) Introduction The AxyPrep Mag FragmentSelect-I purification kit utilizes a unique
More informationHow To Use An Enzymatics Spark Dna Sample Prep Kit For Ion Torrent
SPARK DNA Sample Prep Kit Ion Torrent (SPK0002-V08) Frequently Asked Questions Under what circumstances would I use SPARK DNA Sample Prep Kit for Ion Torrent? Enzymatics SPARK DNA Sample Prep Kit for Ion
More informationA Brief Guide to Interpreting the DNA Sequencing Electropherogram Version 3.0
A Brief Guide to Interpreting the DNA Sequencing Electropherogram Version 3.0 Plant-Microbe Genomics Facility The Ohio State University 484 W.12 th Ave., Columbus, OH 43210 Ph: 614/247-6204 FAX: 614/247-8696
More informationDenaturing Gradient Gel Electrophoresis (DGGE)
Laboratory for Microbial Ecology Department of Earth, Ecological and Environmental Sciences University of Toledo Denaturing Gradient Gel Electrophoresis (DGGE) Background information Denaturing gradient
More informationRNA Extraction and Quantification, Reverse Transcription, and Real-time PCR (q-pcr)
RNA Extraction and Quantification, Reverse Transcription, and Real-time Preparation of Samples Cells: o Remove media and wash cells 2X with cold PBS. (2 ml for 6 well plate or 3 ml for 6cm plate) Keep
More informationReverse Transcription System
TECHNICAL BULLETIN Reverse Transcription System Instruc ons for use of Product A3500 Revised 1/14 TB099 Reverse Transcription System All technical literature is available on the Internet at: www.promega.com/protocols/
More informationLab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity
Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity Today you analyze the results of your bacterial transformation from last week and determine the efficiency
More information7 Electrophoresis. µ proportional to Q
7 Electrophoresis Objectives: A) To perform agarose gel electrophoresis of the proteins isolated in last week's experiment and B) to interpret the banding patterns produced by these proteins. Introduction:
More informationOptimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a
Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a Ryan S. Stowers, 1 Jacqueline A. Callihan, 2 James D. Bryers 2 1 Department of Bioengineering, Clemson University, Clemson,
More informationPowerFecal DNA Isolation Kit
PowerFecal DNA Isolation Kit Catalog No. Quantity 12830-50 50 Preps Instruction Manual Inhibitor Removal Technology (IRT) is a registered trademark of MO BIO Laboratories, Inc. and is covered by the following
More informationPaper Chromatography: Separation and Identification of Five Metal Cations
Paper Chromatography: Separation and Identification of Five Metal Cations Objectives Known and unknown solutions of the metal ions Ag +, Fe 3+, Co 2+, Cu 2+ and Hg 2+ will be analyzed using paper chromatography.
More informationUser Manual. CelluLyser Lysis and cdna Synthesis Kit. Version 1.4 Oct 2012 From cells to cdna in one tube
User Manual CelluLyser Lysis and cdna Synthesis Kit Version 1.4 Oct 2012 From cells to cdna in one tube CelluLyser Lysis and cdna Synthesis Kit Table of contents Introduction 4 Contents 5 Storage 5 Additionally
More informationBiotechnology Explorer. Planning Guide
Biotechnology Explorer Cloning and Sequencing Explorer Series Catalog #166-5000EDU Planning Guide explorer.bio-rad.com Note: This document is for planning purposes only and may vary from the final product
More informationELUTION OF DNA FROM AGAROSE GELS
ELUTION OF DNA FROM AGAROSE GELS OBTECTIVE: To isolate specific bands or regions of agarose-separated DNA for use in subsequent experiments and/or procedures. INTRODUCTION: It is sometimes necessary to
More information