Bacterial 16S rrna V1-3 Amplicon Sequencing Standard Protocol
|
|
- Anthony Long
- 6 years ago
- Views:
Transcription
1 Bacterial 16S rrna V1-3 Amplicon Sequencing Standard Protocol Version 1.05 Skill Prerequisites: DNA handling, gel electrophoresis, DNA concentration measurement, polymerase chain reaction (PCR) Introduction This protocol describes how to make amplicon sequencing libraries of the variable region 1 to 3 (V1-3) of the bacterial 16S rrna gene. The 16S rrna gene is used as a universal phylogenetic marker and for distinguishing and classifying bacteria. Using the 16S rrna V1-3 enables a resolution ranging from family to species level depending on the phylogenetic branches investigated. The library preparation is PCR based and relies on PCR primes targeting conserved sequence regions between the variable regions in the bacteria 16S rrna genes. This means that the analysis will not target bacteria whose sequences deviates from the consensus sequences of these conserved regions. The used primer sequences (F: 5 -AGAGTTTGATCCTGGCTCAG-3 R: 5 -ATTACCGCGGCTGCTGG-3 ) target 78.5% of the known bacteria 16S sequences with sufficient length to be evaluated with 1 or less mismatches (SILVA SSU-114 RefNR database, ). The sequenced part of the amplicon around the 16S rrna gene variable region 1 to 3 starts at E. coli position 28 and ends at E. coli position 516 and is total of 488 bp. However the actual length depends on the target species and might vary up to +/- 75 bp. The amplicon DNA molecule includes the primer sequences and the Illumina sequencing adaptors and is a total of 614 bp when amplified from E. coli and likewise smaller/larger in other species. The protocol assumes the use of 2x300 bp Illumina MiSeq reads to sequence the full V1-3 region. Materials Instruments - Nanodrop1000 (Thermo Scientific) - Electrophoresis chamber and power source - Gel-Doc - Qubit 2.0 Fluorometer (Invitrogen) and/or Infinite M1000 PRO (Tecan) - Thermocycler - MagneSphere Technology Magnetic Separation Stand (Promega) - Tapestation2200 (Agilent) Reagents/consumables - DNAse free tips (10uL, 300uL and 1000 ul) - DNAse free tubes (1.5 ml) Page 1 of 6
2 - 96-well PCR plates (VWR) - PCR strip caps - OptiPlate-96 Black (Perkin Elmer) - Nuclease Free H 2 O (Qiagen) - SeaKem LE Agarose (LONZA) - Gelred (Biotium) - 1kb Generuler - TAE-buffer 50x - Quibit - dsdna BR assay kit (Invitrogen) - QUANT-iT DNA Assay High Sensitivity kit (Invitrogen) - Platinum Taq DNA Polymerase High Fidelity kit (Invitrogen) - dntp mix - Barcoded V1-3 adaptor mixes - Agencourt AMPure XP (Beckman Coulter) - EtOH, 99% - D1K Screentape (Agilent) Method Input sample material IMPORTANT: DNA samples must be extracted using the exact same procedure if they are to be compared. For WWTP Activated Sludge samples and Digester sludge samples use respective standard protocols available on the EB-group Google Drive. Preferably perform DNA extraction of biological triplicates and perform independent amplicon analysis of on each replicate. Sample DNA QC and dilution 1. Quality and Concentration Check a. Nanodrop DNA measurement. Use guidelines provided by the vendor. A260/280 should be 1.8 to 2.0. A260/ to 2.2. Also compare the UV-vis absorbance curve to that of pure DNA. NB: DNA extracted with FastDNA spin kit for soil is known to contain contamination producing a peak around 230 nm. The contaminant does not effect this protocol but may result in overestimation of the DNA concentration. 2. Preliminary dilution 1. Based on the Nanodrop concentrations measurements dilute all samples to 20 ng/ul and in volume of at least 15 ul in a 96-well plate using DNA H 2 O. 3. Run gel electrophoresis (optional) 1. Prepare a 1% agarose gel 1. 1 g agarose, 100 ml 1xTAE-buffer, heat in microwave and gently swirl to completely dissolve agarose. After slight cooling add 2 ul gelred and cast gel. 2. Prepare samples by mixing 1 ul 6x loading buffer, 2 ul sample (20-50 ng) and 3 ul DNA H 2 O. Prepare ladder according to vendor recommendations. 3. Run electrophoresis approximately 80 min at 120V (6 V/cm) 4. Capture gel image on gel-doc, save and analyze. The majority of DNA should be in the range kb. If it is below the DNA is heavily degraded and it might affect the analysis. Page 2 of 6
3 4. Measure DNA concentration of diluted samples (ca. 20 ng/ul) using either Qubit/TECAN 1. Quant-iT HS DNA Assay Kit/Quibit dsdna HS assay kit: Follow standard protocol. Recalibrate instrument with standards every time. Measure samples and standards in duplicate. Load at least 2 ul sample to minimize pipetting error. Re-measure samples where the duplicate values are >10% different from their average. 5. Based on Quant-iT/Quibit measurements dilute to 5 ng/ul with DNA H 2 O. Library PCR 1. Library PCR reaction is run in duplicate for each sample. Remember negative control (DNA H2O) and positive control (sample run before). 2. Prepare master mix: Reagents Final conc. 1 reaction [µl] DNA H2O x10 buffer Platinum High Fidelity x1 2.5 dntp (5 mm) 400 µm 2 MgSO4 (50mM) 1.5 mm 0.75 Platinum Taq DNA Polymerase High Fidelity (0.5 U/µl) 2mU 0.1 Total volume Transfer 21 ul of mastermix to the wells of a 96-well PCR plate. 4. Add 2 ul of assigned barcoded V1-3 adaptor mix (5uM) to each tube. Final conc. is 400 nm. 5. Add 2 ul of template (ca. 10 ng total DNA) 6. Run PCR program 16S AMP PCR V13 30C Temp Time 95 C 2 min 30 cycles 95 C 20 sec 56 C 30 sec 72 C 60 sec 72 C 5 min Page 3 of 6
4 4 C Forever 7. Pool the duplicates. Library QC 1. DNA concentration i. Quant-iT HS DNA Assay Kit/Quibit dsdna HS assay kit: Follow standard protocol. Recalibrate instrument with standards every time. Measure samples and standards in duplicate. Load at least 2 ul sample to minimize pipetting error. Re-measure samples where the duplicate values are >10% different from their average. ii. Remember to measure the concentration of the blank sample. Subtract this reference concentration from all other samples. Use this to detect possible failed PCR reactions. 2. Gel electrophoresis i. Based on the DNA concentrations pick all the potentially failed PCR reactions, 3 good libraries and the negative and positive PCR sample. ii. iii. Library Pooling Run Tapestation 2200 with D1K Screentapes. Follow standard protocol. No replicates. Insure that target amplicon is present (614 bp) and the most abundant product and that no contaminating peaks are present at >400 bp. Depending on the PCR efficiency there can be significant contamination present <400 bp. 1. Pool libraries in equimolar concentrations. The simplest way is to calculate how much sample volume is needed to obtain a specific amount of library. ie. sample concentration is 5 ng/ul and 10 ng library DNA is wanted from each sample = 2 ul sample. Remember another sample might have a concentration of 2.5 ng/ul sample which means 4 ul sample is needed. Library Pool Cleanup 2. Gently shake Agencourt AMPure XP Bottle to resuspend beads, remove required amount and let it equilibrate to room temperature. 3. Transfer 40 ul of bead solution to a 1.5 ml spintube, add 50 ul of library pool to the beads and mix with pipette (10 times up and down). 4. Incubate for 5 minutes at room T. 5. Place sample spintube in magnetic rack for spintubes for 2-4 minutes, until liquid is cleared. 6. Remove liquid with pipette and discard it. 7. Wash bead-pellet with 200 ul 70% EtOH by gently aspirating it over the beads with a pipette. Let it rest for 30s and then remove. Repeat once. NB: EtOH solution should be made fresh every time 8. Ensure no excess liquid is left after the washes. If there is, remove it with a 10 ul pipette. 9. Dry for approximately 10 min to evaporate the EtOH NB: Longer dry times will dry out the pellet and make it difficult to resuspend, resulting in product loss. 10. Remove Place sample spintube from magnetic rack. Add 23 ul of DNA H 2 O water to each well and mix with pipette (>10x up and down) to resuspend beads. 11. Place sample spintube back on the magnetic rack and wait until the liquid clears. 12. Transfer 20 ul of the liquid to a new spintube. Page 4 of 6
5 Library Pool QC 1. DNA concentration (see above) 2. Gel electrophoresis (see above) i. Only the target amplicon (614 bp) should be present on the gel. Literature Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Lozupone, C. A., Turnbaugh, P. J., Knight, R. (2011). Global patterns of 16S rrna diversity at a depth of millions of sequences per sample. Proceedings of the National Academy of Sciences of the United States of America, 108 Suppl, doi: /pnas Caporaso, J. G., Lauber, C. L., Walters, W. a, Berg-Lyons, D., Huntley, J., Fierer, N., Knight, R. (2012). Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. The ISME journal, 6(8), doi: /ismej HMP Consortium - 16S 454 Sequencing Protocol, Version 4.2.2, Effective Date: October 27, 2010 Authors: Jumpstart Consortium Human Microbiome Project Data Generation Working Group Page 5 of 6
6 Revision History V Protocol was created on the basis of numerous journals. V Elaborated on the planning step. Elaborated on DNA input requirements Gel check of DNA quality o Corrected Agarose concentration from 1.0% to 0.5%. o Changed the target DNA molecular size to 3-10 kb. This is the range we see when using our standard DNA extraction protocol (4x40s at 6 m/s bead beating with the FastSPIN DNA kit for soil). Measure DNA Concentration of Diluted samples o Changed replicates from triplicates to duplicates. Our experience have shown that there are little variance on the DNA concentrations measurements performed with primarily the TECAN but also to some degree the Quibit. Furthermore we believe that an exact DNA concentrations is not critical at this point. If it s off <30% it should be okay, but strictly this has not been proven. By reducing from triplicates to duplicates we use less reagents and save time. Mastermix o Corrected dntp stock concentration and final concentration. o Small corrections Library Cleanup o Added 96-well plate ref. number o Small corrections Measure DNA Concentration of libraries o See above Draft Chapter for Storage and reporting o We need to think more about proper naming. V Added prerequisites for using the protocol Introduction o Added an introduction to the protocol, which elaborates a bit on the target region of the 16S. Minor corrections V Minor corrections Cleaned up sample submission description. Added list of materials V Minor corrections Library Cleanup step 2: Changed amount of Ampure bead solution used from 90 ul to 40 ul. This change insures a more stringent removal of unspecific products of up to around 200 bp. At the same time the cost is reduced. V Minor corrections and updates to the introduction. Major changes to library clean up, pooling and validation. Instead of cleaning up all samples and then performing pooling, we perform pooling before cleanup. This should save time and expensive reagents. It should still be possible to detect poor libraries before pooling with the concentration measurement and the gel electrophoresis. Updates to the submission procedure Page 6 of 6
Archaea V3-5 16S rrna Amplicon Sequencing
Archaea V3-5 16S rrna Amplicon Sequencing Standard Protocol Version 1.0 Skill Prerequisites: DNA handling, gel electrophoresis, DNA concentration measurement, polymerase chain reaction (PCR) Introduction
More informationSampling and DNA Extraction from Influent Wastewater Standard Protocol
Sampling and DNA Extraction from Influent Wastewater Standard Protocol Version 7.1 Skill Prerequisites: DNA handling Introduction This protocol explains sampling and DNA extraction from influent wastewater.
More informationHALOPLEX PCR TARGET ENRICHMENT & LIBRARY PREPARATION PROTOCOL. Version 1.0.3, April 2011 For research use only
HALOPLEX PCR TARGET ENRICHMENT & LIBRARY PREPARATION PROTOCOL Version 1.0.3, April 2011 For research use only HALOPLEX PCR TARGET ENRICHMENT & LIBRARY PREPARATION PROTOCOL Halo Genomics AB, 2011 No part
More informationSupplemental File 1: Modified Nextera XT DNA Sample Preparation Guide (Illumina, USA, Part # rev. C, October 2012).
Supplemental File 1: Modified Nextera XT DNA Sample Preparation Guide (Illumina, USA, Part # 15031942 rev. C, October 2012). Required Kit content: Box1 ATM Amplicon Tagment Mix TD Tagment DNA Buffer NPM
More informationBIOO LIFE SCIENCE PRODUCTS. NEXTflex TM 16S V4 Amplicon-Seq Kit 4 (Illumina Compatible) BIOO Scientific Corp V13.01
BIOO LIFE SCIENCE PRODUCTS NEXTflex TM 16S V4 Amplicon-Seq Kit 4 (Illumina Compatible) Catalog #: 4201-01 (16 reactions) BIOO Scientific Corp. 2013 V13.01 TABLE OF CONTENTS GENERAL INFORMATION... 1 Product
More informationFragment Library Preparation
Fragment Library Preparation 5500 Series SOLiD Systems QUICK REFERENCE Note: For safety and biohazard guidelines, refer to the Safety section in the Fragment Library Preparation: 5500 Series SOLiD Systems
More informationTruSeq ChIP Sample Preparation
FOR RESEARCH USE ONLY Date: Illumina Kit Description: NOTE Unless familiar with the protocol in the latest version of the TruSeq ChIP Sample Preparation Guide (part # 15023092), new or less experienced
More informationAmplicon Library Preparation Method Manual. GS FLX Titanium Series October 2009
GS FLX Titanium Series 1. Workflow 3. Procedure The procedure to prepare Amplicon libraries is shown in Figure 1. It consists of a PCR amplification, performed using special Fusion Primers for the Genome
More informationHashimshony, Wagner, Sher & Yanai. CEL-Seq: Single cell RNA-Seq by multiplexed linear amplification (Cell Reports).
CEL-Seq Protocol Hashimshony, Wagner, Sher & Yanai. CEL-Seq: Single cell RNA-Seq by multiplexed linear amplification. 2012 (Cell Reports). Reagents: LoBind tubes 0.5 ml Eppendorf 022431005 Ultra pure RNase
More informationNEXTFLEX 16S V4 Amplicon-Seq Kit (For Illumina Platforms) Catalog #NOVA (Kit contains 8 reactions) Bioo Scientific Corp V18.
NEXTFLEX 16S V4 Amplicon-Seq Kit 2.0-4 (For Illumina Platforms) Catalog #NOVA-4203-01 (Kit contains 8 reactions) Bioo Scientific Corp. 2018 V18.07 This product is for research use only. Not for use in
More informationProcedure & Checklist - Preparing Asymmetric SMRTbell Templates
Procedure & Checklist - Preparing Asymmetric SMRTbell Templates Before You Begin In this procedure, PCR products are generated using two rounds of amplification. The first round uses target specific primers
More informationBIOO LIFE SCIENCE PRODUCTS
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 informationProcedure & Checklist - Preparing SMRTbell Libraries using PacBio Barcoded Universal Primers for Multiplex SMRT Sequencing
Procedure & Checklist - Preparing SMRTbell Libraries using PacBio Barcoded Universal Primers for Multiplex SMRT Sequencing Before You Begin This document describes methods for generating barcoded PCR products
More informationBIOO LIFE SCIENCE PRODUCTS
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 informationPurpose: To sequence 16S microbial DNA isolated from animal fecal matter and perform compositional analysis of bacterial communities.
Purpose: To sequence 16S microbial DNA isolated from animal fecal matter and perform compositional analysis of bacterial communities. Reference: This protocol was adapted from: A custom and streamlined
More informationAmplicon Sequencing Template Preparation
Amplicon Sequencing Template Preparation The DNA sample preparation procedure for Amplicon Sequencing consists of a simple PCR amplification reaction, but uses special Fusion Primers (Figure 1-1). The
More informationNEXTFLEX ChIP-Seq Kit (For Illumina Platforms) Catalog #NOVA (Kit contains 8 reactions) Bioo Scientific Corp V15.
NEXTFLEX ChIP-Seq Kit (For Illumina Platforms) Catalog #NOVA-5143-01 (Kit contains 8 reactions) Bioo Scientific Corp. 2015-2018 V15.07 This product is for research use only. Not for use in diagnostic procedures.
More informationSingle Cell 3 Reagent Kits v2 Quick Reference Cards
Chromium Single Cell 3 Reagent Kits v2 Quick Reference Cards FOR USE WITH Chromium Single Cell 3' Library & Gel Bead Kit v2, 16 rxns PN-120237 Chromium Single Cell 3' Library & Gel Bead Kit, 4 rxns PN-120267
More informationHybridization capture of DNA libraries using xgen Lockdown Probes and Reagents
Hybridization capture of DNA libraries using xgen Lockdown Probes and Reagents For use with: llumina TruSeq adapter ligated libraries xgen Universal Blockers TS Mix (Catalog # 1075474, 1075475, 1075476)
More informationFOR REFERENCE PURPOSES
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 and protocol, so it is important
More informationBIOO LIFE SCIENCE PRODUCTS
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 informationBIOO LIFE SCIENCE PRODUCTS
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 informationBrad s Rapid Ravi for low starting mrna amounts
Brad s Rapid Ravi for low starting mrna amounts Original: Brad Townsley, annotated and updated by Kaisa Kajala (Brady lab) and Mauricio Reynoso (Bailey-Serres lab), latest update 12/8/16. Purpose and Background
More informationGBS v2. A Rieseberg Lab Production. (Pronounced jibs ) (Genotyping-By-Sequencing) Creators: Edd Buckler Lab. Refiner: Greg Baute
A Rieseberg Lab Production January 29, 2013 GBS v2 (Pronounced jibs ) (Genotyping-By-Sequencing) Creators: Edd Buckler Lab Refiner: Greg Baute Perfectors and Writers: Kristin Nurkowski & Gregory Owens
More informationsparq HiFi PCR Master Mix
sparq HiFi PCR Master Mix Cat. No. 95192-050 Size: 50 reactions Store at -25 C to -15 C 95192-250 250 reactions Description The sparq HiFi PCR Master Mix is a high efficiency, high-fidelity, and low bias
More informationThruPLEX -FD Prep Kit Instruction Manual. Single Tube Library Preparation for Illumina NGS Platforms
ThruPLEX -FD Prep Kit Instruction Manual Single Tube Library Preparation for Illumina NGS Platforms Contents Product Description... 2 Kit Contents... 2 Shipping and Storage... 2 Getting Started... 3 Input
More informationATAC-seq Protocol Kaestner Lab
ATAC-seq Protocol Kaestner Lab Reagents 1X PBS Nuclease-free H 2O NP-40 10% (Sigma/Roche, catalog # 11332473001), store at 4 o C Tween-20 10% (Sigma/Roche, catalog # 11332465001), store at 4 o C Digitonin
More informationMultiplexed Strand-specific RNA-Seq Library Preparation for Illumina Sequencing Platforms
Multiplexed Strand-specific RNA-Seq Library Preparation for Illumina Sequencing Platforms Important Things to know before you start: This protocol generates strand-specific reads, but may lead to slightly
More informationChIPmentation CeMM v1.14 (September 2016)
ChIPmentation CeMM v1.14 (September 2016) This protocol works well for efficient histone modification and transcription factor antibodies. For less efficient antibodies sonication different sonication-,
More informationGENERAL INFORMATION...
BIOO LIFE SCIENCE PRODUCTS NEXTflex-96 TM DNA Barcodes (Illumina Compatible) Catalog #: 514106 BIOO Scientific Corp. 2012 V12.11 TABLE OF CONTENTS GENERAL INFORMATION... 1 Product Overview... 1 Contents,
More informationComplete protocol in 110 minutes Enzymatic fragmentation without sonication One-step fragmentation/tagging to save time
Molecular Cloning Laboratories Manual Version 1.2 Product name: MCNext UT DNA Sample Prep Kit Cat #: MCUDS-4, MCUDS-24, MCUDS-96 Description: This protocol explains how to prepare up to 96 pooled indexed
More informationUpdated August well High-multiplexing Tagmentation and Amplification Robyn Tanny August Company Kit Catalog Number.
96-well High-multiplexing Tagmentation and Amplification Robyn Tanny August 2015 This protocol uses the following purchased reagents: Company Kit Catalog Number Illumina Nextera DNA Sample Preparation
More informationBacterial Iso-Seq Transcript Sequencing Using the SMARTer PCR cdna Synthesis Kit and BluePippin Size-Selection System
Please note: the unsupported protocols described herein may not have been validated by Pacific Biosciences and are provided as-is and without any warranty. Use of these protocols is offered to those customers
More informationUser-Demonstrated Protocol: BD Single-Cell Multiplexing Kit Human
User-Demonstrated Protocol: BD Single-Cell Multiplexing Kit For use with the 10x Chromium Single Cell 3 Reagent Kit v2 01/2018 Doc ID: 179682 Rev. 1.0 Contents Disclaimer on page 3 Overview on page 3 Workflow
More informationGuidelines for Preparing 20 kb SMRTbell Templates
Guidelines for Preparing 20 kb SMRTbell Templates User Bulletin This Bulletin provides recommendations and tips for preparing 20 kb SMRTbell templates using the BluePippin size-selection method. Once you
More informationNEXTFLEX Rapid Directional RNA-Seq Kit (For Illumina Platforms) Catalog #NOVA (Kit contains 8 reactions)
NEXTFLEX Rapid Directional RNA-Seq Kit (For Illumina Platforms) Catalog #NOVA-5138-07 (Kit contains 8 reactions) Bioo Scientific Corp. 2014-2018 V18.07 This product is for research use only. Not for use
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 informationGenome Reagent Kits v2 Quick Reference Cards
Chromium Genome Reagent Kits v2 Quick Reference Cards FOR USE WITH Chromium Genome Library Kit & Gel Bead Kit v2, 16 rxns PN-120258 Chromium Genome Chip Kit v2, 48 rxns PN-120257 Chromium i7 Multiplex
More informationFunctional Genomics Research Stream. Research Meeting: November 8, 2011 cdna Library Construction for RNA-Seq
Functional Genomics Research Stream Research Meeting: November 8, 2011 cdna Library Construction for RNA-Seq Lab Issues Don t leave out boxes of water + ethidium bromide Minimize tip boxes in phenol waste
More informationFragment Library Preparation Using the AB Library Builder System
Fragment Library Preparation Using the AB Library Builder System 5500 Series SOLiD Systems QUICK REFERENCE Note: For safety and biohazard guidelines, refer to the Safety section in the Fragment Library
More informationIllumina Sequencing Sample Preparation for use with CRISPRi/a-v2 Libraries
Illumina Sequencing Sample Preparation for use with CRISPRi/a-v2 Libraries Overview This protocol describes the four steps required for generating sequencing samples from cells harvested from screens conducted
More informationNxSeq Long Mate Pair Library Kit
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) 831-9011 FAX: (608) 831-9012 lucigen@lucigen.com www.lucigen.com
More informationSingle Cell 3 Reagent Kits v2 Quick Reference Cards
Chromium Single Cell 3 Reagent Kits v2 Quick Reference Cards FOR USE WITH Chromium Single Cell 3' Library & Gel Bead Kit v2 PN-120237 Chromium Single Cell 3 Chip Kit v2 PN-120236 Chromium i7 Multiplex
More informationNEBNext. Ultra II RNA Library Prep Kit for Illumina
LIBRARY PREPARATION NEBNext Ultra II RNA Library Prep Kit for Illumina Instruction Manual NEB #E7770S/L, #E7775S/L 24/96 reactions Version 1.0 4/17 be INSPIRED drive DISCOVERY stay GENUINE This product
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 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 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 informationGENERAL INFORMATION...
BIOO LIFE SCIENCE PRODUCTS NEXTflex TM DNA Barcodes - 6 (Illumina Compatible) Catalog #: 514101 (48 reactions) BIOO Scientific Corp. 2012 V12.11 TABLE OF CONTENTS GENERAL INFORMATION... 1 Product Overview...
More informationPremium WGBS Kit. Whole Genome Bisulfite Sequencing. Cat. No. C (8 rxns) Version 1 I 07.15
Premium WGBS Kit Whole Genome Bisulfite Sequencing Cat. No. C02030034 (8 rxns) Version 1 I 07.15 Contacts diagenode headquarters Diagenode s.a. BELGIUM EUROPE LIEGE SCIENCE PARK Rue Bois Saint-Jean, 3
More informationThermo Scientific MuSeek Library Preparation Kit for Ion Torrent
PRODUCT INFORMATION Thermo Scientific MuSeek Library Preparation Kit for Ion Torrent Cat. no. 4480829 For 10 rxns Lot Exp. Store below -70 C before opening For barcoded DNA fragment library generation
More informationLow cost and non-toxic genomic DNA extraction for use in molecular marker studies.
Low cost and non-toxic genomic DNA extraction for use in molecular marker studies. Version 1.4, February 28 th, 2013. Prepared by Bernhard Hofinger, Owen Huynh and Brad Till. 1. OBJECTIVE To develop and
More informationReduced representation bisulfite sequencing Updated 5/19/16, R Kartzinel + K Brzeski Updated 9/12/16, R Kartzinel
Reduced representation bisulfite sequencing Updated 5/19/16, R Kartzinel + K Brzeski Updated 9/12/16, R Kartzinel Notes: This protocol is based on the library preparation protocols in the NEBNext Multiplex
More informationQuick-16S NGS Library Prep Kit Catalog No. D6400 Quick Protocol: High Microbial DNA Samples
Notice The optimal DNA concentration for the Quick-16S NGS Library Prep Kit is 5-20 ng/µl bacterial DNA, excluding host DNA. This Quick Protocol should be used only for DNA samples that are concentrated
More informationSupplementary Protocol: CIRCLE-seq Library Preparation
Supplementary Protocol: CIRCLE-seq Library Preparation Reagent Gentra Puregene Tissue Kit Qubit dsdna BR Assay Kit Agencourt AMPure XP magnetic beads High throughput, with bead, PCR-free Library Preparation
More informationNEXTflex Cystic Fibrosis Amplicon Panel. (For Illumina Platforms) Catalog # (Kit contains 8 reactions) Bioo Scientific Corp V17.
NEXTflex Cystic Fibrosis Amplicon Panel (For Illumina Platforms) Catalog #4231-01 (Kit contains 8 reactions) Bioo Scientific Corp. 2017 V17.02 This product is for research use only. Not for use in diagnostic
More informationJetSeq Flex DNA Library Preparation Kit. Product Manual
JetSeq Flex DNA Library Preparation Kit Product Manual 2 Product Manual bioline.com/jetseq JetSeq Flex DNA Library Preparation Kit JetSeq Flex DNA Library Preparation Kit TABLE OF CONTENTS 1 Kit contents
More informationSureSeq NGS Library Preparation Kit Handbook
SureSeq NGS Library Preparation Kit Handbook Catalogue Number: 500070 (16 reactions) 500073 (48 reactions) Oxford Gene Technology Founded by Professor Ed Southern, Oxford Gene Technology (OGT) brings world-class
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 informationComparison of ExoSAP-IT and ExoSAP-IT Express reagents to alternative PCR cleanup methods
WHITE PAPER ExoSAP-IT PCR cleanup reagents Comparison of ExoSAP-IT and ExoSAP-IT Express reagents to alternative PCR cleanup methods Abstract Here we present superior workflow advantages of enzymatic PCR
More informationFragment Library Preparation
USER GUIDE Fragment Library Preparation 5500 Series SOLiD Systems Publication Part Number 4460960 Rev. B prepare libraries prepare beads run sequencer analyze data For Research Use Only. Not intended for
More informationCell Hashing Protocol
Cell Hashing Protocol For experiments involving Cell Hashing, use cost per cell calculator to plan experiments, determine number of hashes, number of cells to load, expected doublet rates (detected and
More informationCITE-seq & Cell Hashing Protocol
CITE-seq & Cell Hashing Protocol For experiments involving Cell Hashing, use cost per cell calculator to plan experiments, determine number of hashes, number of cells to load, expected doublet rates (detected
More informationPrepare a Barcoded Fragment Library with the SOLiD Fragment Library Barcoding Kit 1 96
QUICK REFERENCE CARD Prepare a Barcoded Fragment Library with the SOLiD Fragment Library Barcoding Kit 1 96 Note: For safety and biohazard guidelines, refer to the Safety section in the Applied Biosystems
More informationPreparation of Reduced Representation Bisulfite Sequencing (RRBS) libraries
Preparation of Reduced Representation Bisulfite Sequencing (RRBS) libraries Ambre Bender and Michael Weber CNRS University of Strasbourg UMR7242 Biotechnology and Cell signalling 300, Bd Sébastien Brant
More informationJoint RuminOmics/Rumen Microbial Genomics Network Workshop
Joint RuminOmics/Rumen Microbial Genomics Network Workshop Microbiome analysis - Amplicon sequencing Dr. Sinéad Waters Animal and Bioscience Research Department, Teagasc Grange, Ireland Prof. Leluo Guan
More informationirepertoire Inc. For Research Use Only, Not to be Used for Clinical Diagnostics.
For Research Use Only, Not to be Used for Clinical Diagnostics. v102117 The ir logo is a registered trademark of irepertoire, Inc. irepertoire is a trademark of irepertoire, Inc. Illumina, HiSeq,and MiSeq,
More informationMastermix 16S Complete, DNA-free
Mastermix 16S Complete, DNA-free For the PCR detection and identification of bacteria using universal 16S rdna primers For research use only Cat. No. S-020-0100 Cat. No. S-020-0250 Cat. No. S-020-1000
More information3.1 RNA Fragmentation, Priming and First Strand cdna Synthesis. 3.1A RNA Fragmentation and Priming Starting from Intact or Partially Degraded RNA:
CHAPTER 3 Please refer to revision history for a summary of protocol updates Symbols SAFE STOP This is a point where you can safely stop the protocol and store the samples prior to proceeding to the next
More informationProtocol for submitting gdna for Illumina 16S rrna gene sequencing, V4 region
Protocol for submitting gdna for Illumina 16S rrna gene sequencing, V4 region Version: 2018 Summary: This protocol is for the submission of gdna to generate libraries for 16S rrna sequencing, which can
More informationQUANTITATIVE RT-PCR PROTOCOL (SYBR Green I) (Last Revised: April, 2007)
QUANTITATIVE RT-PCR PROTOCOL (SYBR Green I) (Last Revised: April, 007) Please contact Center for Plant Genomics (CPG) facility manager Hailing Jin (hljin@iastate.edu) regarding questions or corrections.
More informationCleanup. Total Time 2.5 hr
sparq DNA Library Prep Kit Cat. No. 95191-024 Size: 24 reactions Store at -25 C to -15 C 95191-096 96 reactions Description The sparq DNA Library Prep Kit provides components for the rapid construction
More informationIon AmpliSeq Library Kit 2.0
QUICK REFERENCE Ion AmpliSeq Library Kit 2.0 DNA Library Preparation with 1- or 2-Pool Panels Using qpcr Quantification Catalog Numbers 4475345, 4480441, 4480442, 4479790, A31133, A31136, A29751 Pub. No.
More informationKAPA Library Preparation Kits
Technical Data Sheet KAPA Library Preparation Kits Illumina series Product Description The KAPA Library Preparation Kit provides all of the enzymes and reaction buffers required for constructing libraries
More informationIon TrueMate Library Preparation
USER GUIDE Ion TrueMate Library Preparation for use with: Ion TrueMate Library Kit Ion TrueMate Plus Library Kit Catalog Numbers A25614 and A25656 Publication Number MAN0010280 Revision B.0 For Research
More informationIon Total RNA-Seq Kit v2
Ion Total RNA-Seq Kit v2 USER GUIDE for use with: Ion PGM System Ion Proton System Ion S5 System Ion S5 XL System Catalog Numbers 4475936, 4479789, 4475485 Publication Number MAN0010654 Revision C.0 For
More informationsparq DNA Frag & Library Prep Kit
sparq DNA Frag & Library Prep Kit Cat. No. 95194-024 Size: 24 reactions Store at -25 C to -15 C 95194-096 96 reactions Description The sparq DNA Frag & Library Prep Kit provides reagents essential for
More informationHEAT-Seq and HEAT-Seq Ultra Target Enrichment User s Guide Version 1.1
HEAT-Seq and HEAT-Seq Ultra Target Enrichment User s Guide Version 1.1 For Research Use Only. Not for use in diagnostic procedures. Copyright 2016-2017 Roche Sequencing Solutions, Inc. All Rights Reserved.
More informationNEBNext Direct Custom Ready Panels
LIBRARY PREPARATION NEBNext Direct Custom Ready Panels Instruction Manual NEB #E6631S/L/X 8/24/96 reactions Version 1.0 4/18 be INSPIRED drive DISCOVERY stay GENUINE i This product is intended for research
More informationCOFACTOR GENOMICS Cofactor ImmunoPrism Kit Version 1.0 THE LEADERS IN RNA. Cofactor ImmunoPrism Kit Version 1.0
THE LEADERS IN RNA Cofactor ImmunoPrism Kit Version 1.0 Table of Contents Introduction Procedure Protocol Notes Thermal Cycler Programs Optional Protocol Modifications Support Protocol Overview Kit Reagents
More informationrrna subtraction protocol for metatranscriptomics
rrna subtraction protocol for metatranscriptomics Recommended materials/kits Price Vendor Stock Number MEGAscript transcription kit $249 Ambion AM1334 MEGAclear TM kit $89 Ambion AM1908 RNeasy MinElute
More informationXactEdit Cas9 Nuclease with NLS User Manual
XactEdit Cas9 Nuclease with NLS User Manual An RNA-guided recombinant endonuclease for efficient targeted DNA cleavage Catalog Numbers CE1000-50K, CE1000-50, CE1000-250, CE1001-250, CE1001-1000 Table of
More informationMeDIP-seq library construction protocol v2 Costello Lab June Notes:
MeDIP-seq library construction protocol v2 Costello Lab June 2010 Notes: A. For all Qiagen gel extraction steps (Qiaquick and MinElute), melt gel slice at 37º C instead of 50º (see Quail et al 2008 Nature
More informationLow-cost DNA extraction for use in TILLING and Ecotilling assays
Low-cost DNA extraction for use in TILLING and Ecotilling assays Version 1.3 (January 31, 2013) Plant Breeding and Genetics Laboratory Prepared by Bernhard Hofinger and Bradley Till 1. MATERIALS Company
More informationArcher ALK, RET, ROS1 Fusion Detection v1 Illumina Platform
Archer ALK, RET, ROS1 Fusion Detection v1 Illumina Platform P/N AK0001-8 Archer ALK, RET, ROS1 Fusion Detection v1 for Illumina Platform IFU-AK0001-8 Rev. A Table of Contents Product Description..3 Workflow
More informationSureSeq Ovarian Cancer Panel Handbook
SureSeq Ovarian Cancer Panel Handbook Catalogue Numbers: 600074 (96 reactions) 600073 (16 reactions) Oxford Gene Technology Founded by Professor Ed Southern, Oxford Gene Technology (OGT) world-class genetics
More information5X WGS Fragmentation Mix
4.1. 5X WGS Fragmentation Mix Instructions for Use Product Number Y9410L and Y9410F Product Description The 5X WGS Fragmentation Mix is an enzyme mix to perform DNA fragmentation, end-repair and da-tailing
More informationMethods S1. Minimal Starting Amount Sample Preparation Protocol (MSA-Cap)
1 Methods S1 Minimal Starting Amount Sample Preparation Protocol (MSA-Cap) 1. Methods. 1.1. Fragmentation. Start from 50-60 ng DNA in 10-30 µl Elution buffer (EB) or TE buffer (10mM Tris, ph 7.5/ 1 mm
More informationCATS RNA-seq v2 library preparation protocol on RNA from FFPE-samples
CATS RNA-seq v2 library preparation protocol on RNA from FFPE-samples INTRODUCTION The following protocol offers a streamlined solution for whole transcriptome sequencing studies from human/ mouse/rat
More informationNEBNext. for Ion Torrent LIBRARY PREPARATION KITS
NEBNext for Ion Torrent LIBRARY PREPARATION KITS NEBNEXT PRODUCTS FOR ION TORRENT Table of Contents 3 General Introduction 4 5 6 6 7 8 DNA Library Preparation Workflow Product Selection Product Details
More informationNEBNext FFPE DNA Repair Mix
LIBRARY PREPARATION NEBNext FFPE DNA Repair Mix Instruction Manual NEB #M6630S/L 24/96 reactions Version 4.0 4/18 be INSPIRED drive DISCOVERY stay GENUINE This product is intended for research purposes
More informationTotal RNA Sample QC. PCR 8-tube strip USA Scientific
Total RNA Sample QC Version Number: 2.0 Version Date: 10/1/2018 Authors: Mansi Chovatia, Timothy Williams, Aditi Sharma, Alba Guiterrez, Juying Yan, Kathleen Lail, Chia-Lin Wei and Yuko Yoshinaga SUMMARY
More informationEbola virus sequencing protocol
Ebola virus sequencing protocol Nanopore amplicon native barcoding Document: ARTIC-EBOV-seqSOP-v1.0.0 Creation Date: 2018-05-17 Revision Date: 2018-05-27 Forked from: doi:10.1038/nprot.2017.066 Author:
More informationTwist Human Core Exome EF Multiplex Complete Kit, 16 Samples PN
Twist Human Core Exome Enrichment Kit Twist Human Core Exome EF Multiplex Complete Kit, 16 Samples PN 100252 Reagents for preparing 16 exome-enriched libraries ready for sequencing from human genomic DNA
More informationBiotool DNA library prep kit V2 for Illumina
Biotool DNA library prep kit V2 for Illumina Description Biotool DNA library prep kit V2 for Illumina is developed specially for the Illumina high-throughput sequencing platform, and generates sequencing-ready
More informationNEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina
LIBRARY PREPARATION NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina Instruction Manual NEB #E6420S/L 24/96 reactions Version 1.0 4/18 be INSPIRED drive DISCOVERY stay GENUINE i This product
More informationLibrary Loading Bead Kit (EXP-LLB001) Agencourt AMPure XP beads Vortex mixer. Freshly prepared 70% ethanol in nucleasefree
Before start checklist Materials Consumables Equipment Native Barcoding Kit 1D (EXP-NBD103) NEBNext End repair / da-tailing Module (E7546) Thermal cycler at 20 C and 65 C Ligation Sequencing Kit 1D ( NEB
More informationRen Lab ENCODE in situ HiC Protocol for Tissue
Ren Lab ENCODE in situ HiC Protocol for Tissue Pulverization, Crosslinking of Tissue Note: Ensure the samples are kept frozen on dry ice throughout pulverization. 1. Pour liquid nitrogen into a mortar
More informationLab Book igem Stockholm Sortase A. Week 5
Sortase A Week 5 Summarized below are the experiments conducted this week in chronological order. Click on the experiment name to view it. To go back to this summary, click Summary in the footer. Summary
More informationKAPA Library Preparation Kit Ion Torrent Platforms
KAPA Library Preparation Kit KR0573 v2.16 This provides product information and a detailed protocol for the KAPA Library Preparation Kit for Ion Torrent platforms. Contents Product Description...2 Product
More informationKAPA Library Preparation Kit Ion Torrent Platforms
KR0573 - v1.13 Product Description This is designed for the preparation of libraries for sequencing on the Ion Personal Genome Machine (PGM ) and Ion Proton semiconductor sequencers. The kit provides all
More information