Next-Generation Sequencing Strategies Enable Routine. Detection of Balanced Chromosome Rearrangements. for Clinical Diagnostics and Genetic Research
|
|
- Christopher Pope
- 7 years ago
- Views:
Transcription
1 Supplemental Data American Journal of Human Genetics, Volume 88 Next-Generation Sequencing Strategies Enable Routine Detection of Balanced Chromosome Rearrangements for Clinical Diagnostics and Genetic Research Michael E. Talkowski, Carl Ernst, Adrian Heilbut, Colby Chiang, Carrie Hanscom, Amelia Lindgren, Andrew Kirby, Shangtao Liu, Bhavana Muddukrishna, Toshiro K. Ohsumi, Yiping Shen, Mark Borowsky, Mark J. Daly, Cynthia C. Morton, and James F. Gusella
2 Table S1. Captured regions and alignment statistics Subject Region Chr Length (bp) Mean Coverage Repeat Masked Mean Coverage Unmasked % masked % Masked Aligned % Unmasked Aligned % No Coverage (Masked) % No Coverage (Unmasked) Subject % 83.6% 96.3% 12.2% 3.7% Subject % 88.5% 93.2% 10.6% 6.8% Subject % 9.3% 3.9% 90.7% 96.1% Subject % 94.5% 100.0% 2.8% 0.0% Subject % 91.3% 99.1% 7.2% 0.6% Subject % 73.3% 84.1% 20.4% 15.9% Subject % 94.5% 100.0% 3.3% 0.0% Subject % 97.0% 99.7% 2.4% 0.3% Subject % 91.0% 99.8% 4.8% 0.2% Subject % 93.6% 98.6% 2.7% 1.4% Subject % 94.9% 98.9% 3.7% 1.1% Results for capture and alignment performance for each region provided. As discussed, the majority of bases within the chr 5 region of Subject 8 could not be uniquely aligned. Capture statistics were calculated from all other regions. Masked refers to regions designated as repetitive by RepeatMasker. Overall, 40.4% of reads mapped to the targeted regions (2,528,280 total bases captured), with an average read depth of 42 reads per base within the targeted regions and reads per base in non-targeted regions. Our analysis suggests 90.7% of repeat-masked bases in targeted regions were theoretically mappable by 75 bp reads, roughly corresponding to our estimated genome-wide mappability rate of 90.3%.
3 Figure S1. Sequencing results for Subject 3 shows five reads supporting an inversion of chromosome 5 (46,XX,inv(5)(p14.2q14.3)) derived from a single lane of Illumina GAIIx sequencing using the Illumina mate-pair protocol (Illumina, Inc). By contrast, a translocation between chromosomes 3 and 18 was easily delineated from thirty-two supporting reads using our EcoP15I custom barcoded jumping libraries.
4 Figure S2. Despite the low breakpoint coverage, PCR and FISH analysis of Subject 3 confirms the presence of a pericentric inversion of chromosome 5 with breakpoints localized to 5p14.2 and 5q14.3.
5
6
7
8
9
10
11 Figure S3. Capture performance of each subject shown in the UCSC browser. The Y-axis read depth is set at 10X, not the actual read depth for a given base (average depth per base was 42x). Subject number and region is shown to the left of the Y-axis. CRG 75 bp alignability data and UCSC Repeat- Masker track is shown below the alignability data. Targeted Capture Library Protocols Samples were processed using the NEBNext DNA Sample Prep Master Mix Set 1 (New England Biolabs, Inc.) sample preparation kits described above, custom PCR primers for Solexa technology described previously 9, and Illumina adapter oligos following the Illumina standard paired-end library formation procedures described above with the exception of additional PCR steps prior to and following each hybridization step, per manufacturer s instructions (Agilent Technologies, Inc.). Chips were processed according to manufacturer s instructions using commercially supplied reagents. To increase specificity based on previous studies 11, we included 50 ul of C o T1 (Invitrogen, Inc.), 50 ul blocking buffer, and 5 ul each of sense and antisense blocking oligos to Illumina specific adaptors (200 um). Eluate from the initial hybridization was extracted, concentrated to 50 ul, and eight 18 cycle PCR reactions were performed using 5 ul aliquots of DNA. Purified DNA was then re-applied to the chip for a second hybridization, a technique previously found to increase sensitivity and specificity of the experiment 5, followed by another round of PCR. Approximately 5% of each captured library was cloned using a pcr-blunt II-TOPO vector. DNA was extracted for colonies and Sanger sequenced to determined capture specificity (Zero Blunt TOPO PCR Cloning Kit, Invitrogen, Inc.).
12 Jumping Library Protocol and Reagent List Hydroshear R from Genomic Solutions Cat# X Low TE buffer Qiaquick PCR purification kit from Qiagen Cat# MinElute reaction clean up kit from Qiagen Cat# Qiaquick R gel purificaiton kit from Qiagen Cat# Agilent lab chip kit from Agilent Cat# End-It TM DNA End-Repair Kit Epicentre Cat# ER0720 Cap adaptor oligos(hplc purified): 5 Phos/ACAGCAG 3 3 ACTGTCTGC/Phos 5 Quick Ligation kit from NEB Cat#: M2200s Nuclease-free water from Ambion Cat#: AM9930 Seakem LE Agarose from Lonza Cat# kb ladder from NEB Cat#3272S SYBR Gold nucleic acid gel stain from Invitrogen Cat# S11494 Internal adaptor oligos (HPLC purified); T*=biotin labeled position 5 Phos/CGTACAT*CCGCCTTGGCCGT 3 3 TGGCATGTAGGCGGAACCGG/Phos 5 ATP (25mM) 10X Plasmid-safe buffer Plasmid-safe DNase (10U/ul) 100X BSA from NEB Cat# B9001S EcoP15I, 10U/ul from NEB Cat# R0646L Sinefungin from Sigma Cat# S8559 Buffer 3 from NEB Cat# Klenow DNA polymerase from NEB Cat# M0210S Tris-HCl 500mM NaCl 5M EDTA 500mM Triton X-100 Tween 20 Streptavidin Myone C1Beads from Invitrogen Cat# D
13 NEBNext da-tailing Module: NEB (Cat# E6053s) 2X Phusion mastermix NuSieve 3:1 Agarose from Lonza Cat# TrackIt 25bp ladder from Invitrogen Cat# Methods based on Applied Biosystems Mate-Pair library formation method (Applied Biosystems, Inc.), with modifications listed. 1. Fragment genomic DNA by Hydroshear or Covaris Equipment and Reagents: 1X Low TE buffer Qiaquick PCR purification kit from Qiagen MinElute reaction clean up kit from Qiagen Agilent labchip kit from Agilent Process: Shear ug of genomic DNA (gdna) diluted with 1X low TE to desired fragments (~ kb in this study) according to manufacturer s instructions on the Hydroshear or Covaris (Covaris used in these studies). Collect the sheared gdna sample and perform Qiagen cleanup following the manufacturer s manual, elute with total 203ul EB buffer. Perform QC with an Agilent Bioanalyzer. 2. End Repair Reagents: End-It TM DNA End-Repair Kit Epicentre MinElute reaction clean up kit from Qiagen a) Combine and mix the following components in a microcentrifuge tube: Component Volume(ul) Sheared DNA X End-It Buffer 30 End-It ATP(10mM) 30
14 End-It dntps(2.5mm) 30 End-It Enzyme Mix 10 Total 300 Set up 50ul reaction per 5ug DNA material (protocol here assuming 30ug gdna starting material) b) Incubate the mixture at room temperature for 30 mins. c) Clean up the reaction with Qiagen kit and elute with100ul EB buffer. d) Quantify the DNA with Pico assay or Nanodrop. 3. EcoP15I Cap adaptors ligation Reagents: Cap adaptor oligos( from IDT, HPLC purified:50um): 5 Phos/CTGCTGT AC 3 3 GACGACA/Phos 5 Quick Ligation kit from NEB Nuclease-free water from Ambion MinElute reaction clean up kit from Qiagen a) Combine and mix the following components in a microcentrifuge tube: Component Volume(ul) Cap adaptor(ds) 50uM Y 2X Quick Ligase Buffer 150 Quick Ligase 7.5 DNA 100 Nuclease free H 2 O Variable Total 300 Keep the fragment to cap adaptor molecule ratio 1:100. Xpmol DNA= (ug DNA)*10^6/660 / average fragment length(bp) Y(ul )adapter needed= 100*X/50
15 b) Incubate the mixture at room temperature for 10mins. c) Clean up the reaction with Qiagen kit and elute with102ul EB buffer. 4. Gel-size Selection Reagents: Seakem LE Agarose from Lonza Qiaquick gel purificaiton kit from Qiagen 1kb ladder from NEB SYBR Gold nucleic acid gel stain from Invitrogen a) Prepare a 0.8% agarose gel in 1XTAE buffer b) Load all materials from step 3. Use multiple lanes if necessary. Include a lane with 1 kb or 1 kb Plus ladder. c) Run the gel at 120v until the yellow dye in the marker is close to the bottom of the gel. d) Stain the gel with SYBR for 30mins. e) Excise the desired insert size range of the gel with a clean razor blade. f) Process gel purification following the manufacturer s instructions (dissolve the gel piece in QG buffer at room temperature). g) Quantify Pico Green and Agilent Bioanalyzer assay. 5. DNA Circularization and DNase Treatment Reagents: Internal adaptor oligos (from IDT, HPLC purified: 2uM) Positions in fuschia can be used for index sequence T*=biotin labeled position 5 Phos/CGTACAT*CCGCCTTGGCC GT 3 3 TGGCATGTAGGCGGAACCGG/Phos 5 T*=biotin labeled position Quick Ligation kit from NEB Nuclease-free Water from Ambion MinElute reaction clean up kit from Qiagen Dilute of ligation reaction to achieve intra-molecular ligation J=63.4/ (DNA size in kb) 1/2 I= (J/0.95 J) =ug/ml for dilution of ligation reaction
16 I is the desired final [DNA] in the circularization reaction in a) below: a) Combine and mix the following components in a micro centrifuge tube: Component Volume(ul) Internal adaptor(ds) 2uM X 2X Quick Ligase Buffer 250 Quick Ligase 12.5 DNA variable Total (use Nuclease-free H 2 O) 500 Keep the fragment to internal adaptor molecule ratio 1:3; Circularize all material from last step, set up more reactions as necessary and keep the DNA dilution of ligation reaction. X(ul)=1.5*10^6*(DNA-ug)/660/fragment length. b) Incubate the mixture at room temperature for 10mins. c) Clean up the reaction with Qiagen kit and elute with 60ul EB buffer total. d) Combine and mix the following components: Component Volume(ul) ATP (25mM) 5 10X Plasmid-safe buffer 10 Plasmid-safe DNase (10U/ul) Y DNA 60 Nuclease-free H 2 O Variable Total 100 1ul DNase enzyme will be needed per 3ug DNA material. e) Incubate the reaction at 37 0 C for 40mins. f) Clean up the reaction with Qiagen kit and elute in 30ul EB buffer. 6. Digest the circularized DNA with EcoP15I and End repair Reagents: 100X BSA from NEB EcoP15I kit, 10U/ul from NEB
17 Sinefungin from Sigma Klenow DNA polymerase from NEB a) Combine and mix the following components b) Incubate at 37 o C overnight (16 hour minimum). c) Add 1ul of 10mM Sinefungin, 2ul of 10XATP and 0.5ul of EcoP15I to the 100ul reaction (103.5ul total). d) Incubate the reaction mixture at 37 o C for 1 hour. e) Denature the enzyme at 65 o C for 20mins and chill on ice for 5 mins. f) Perform end repair in the same tube to conserve sample. Add 1.5ul of dntp mix (25mM each) and 1ul Klenow large fragment to the 103.5ul reaction (106ul total). g) Incubate the reaction at room temperature for 30mins. h) Denature the enzyme at 65 o C for 20 mins and chill on ice for 5 mins. i) Add 200ul 2X streptavidin binding buffer and 94ul nuclease free H 2 O to the reaction (400ul total). Component Volume(ul) Circularized DNA 30 10X Buffer XBSA 10 Sinefungin(10mM) 1 10X ATP(25mM) 20 EcoP15I (10U/ul) X Nuclease-free H 2 O variable Total ul to 5ul EcoP15I enzyme needed per ug DNA (2-6kb). 7. Bind the library molecules to Streptavidin beads Reagents: Tris-HCl 500mM NaCl 5M EDTA 500mM Triton X-100 Tween 20 Streptavidin Myone C1 Beads from Invitrogen a) Prepare the 2X streptavidin bead binding buffer
18 Components Volume(ul) Tris-HCl 500mM PH= NaCl 5M 200 EDTA 500mM 1 Nuclease free H 2 O 289 Total 500 b) Prepare 1X BSA solution Component Volume(ul) 100X BSA 5 Nuclease free H 2 O 495 Total 500 c) Prepare 1X Bead Wash buffer Components Volume(ul) Triton X Tween EDTA 500mM 20 Nuclease free H 2 O 940 Total 1000 d) Vortex the bottle of Dynal MyOne C1 streptavidin beads and transfer 90ul into a lowbind tube. e) Add 500ul 1X bead wash buffer and vortex for 15 seconds, then pulse-spin. f) Place the tube in a magnetic rack for at least 1min. After the solution clears, discard the supernatant. g) Add 500ul of 1XBSA and vortex for 15 seconds, then pulse-spin. h) Place the tube in a magnetic rack for at least 1 min. After the solution clears, discard the supernatant. i) Add 500ul 1X bead binding buffer and vortex for 15 seconds, then pulse-spin/ j) Place the tube in a magnetic rack for at least 1 min. After the solution clears, discard the supernatant k) Add the entire 400ul solution of library molecules in Streptavdin bead binding buffer to the pre-washed beads and vortex.
19 l) Mix by rotation at room temperature for 30 mins to 1 hour, then pulse-spin. m) Place the tube in a magnetic rack for at least 1 min. After the solution clears, discard the supernatant. n) Resuspend the beads in 500ul of 1XBead Wash buffer and vortex for 15 seconds, then pulse-spin. o) Place the tube in a magnetic rack for at least 1min. After the solution clears, discard the supernatant. p) Resuspend the beads in 500ul of 1X bead wash buffer and transfer beads to a new Lowbind tube. Vortex for 15 seconds, then pulse-spin. q) Place the tube in a magnetic rack for at least 1min. After the solution clears, discard the supernatant. r) Resuspend the beads in 500ul of 1X da tailing reaction buffer and transfer beads to a new Lowbind tube. Vortex for 15 seconds, then pulse-spin. s) Place the tube in a magnetic rack for at least 1min. After the solution clears, discard the supernatant. t) Set up da tailing reaction immediately as follows. 8. da tailing, Illumina adaptor ligation and PCR enrichment Reagents: NEBNext da-tailing Module Quick Ligation kit: NEB Adapter: ligate Illumina PE adapters or custom designed adapters from Bentley et al., 2008 (Bentley et al., Nature, 2008 Nov 6; 456(7218):53-9. PMID: Component Volume (ul) DNA On beads 10X reaction buffer 5ul Klenow (exo - ) 3ul H 2 O 42ul Total 50ul Incubate the reaction on rotator at 37 o C for 30mins. Clean up the reaction following steps m) to t) of section 7 above with the following modifications: Step r): 500ul 1X Quick ligation buffer instead of 1X da tailing buffer; Step t): set up adaptor ligation instead of da tailing Adapter ligation Component Volume (ul)
20 DNA On beads 2X ligation buffer 25ul Quick ligase 2.5ul Adapters(15uM) 10ul H2O 12.5ul Total 50ul Incubate the reaction on rotator at room temperature for 10mins. Clean up the reaction following steps m) to t) of part 7 above with these modifications. Step r): 500ul EB buffer instead of 1X Quick ligase buffer. Step t): Resuspend the beads in 30ul EB buffer PCR enrich library molecules Component Volume (ul) DNA 10ul Primer 1 1ul Primer 2 1ul 2X Phusion mastermix 25ul H2O 13ul Total 50ul Amplify all 30ul DNA material to get high complexity library with fewer PCR cycles. Usually 8 to 12 cycles is enough to get PCR product dependent on starting material and how much loss during the whole process. PCR Program (98 o C 30s; 98 o C 10s, 65 o C 30s, 72 o C 30s; repeat 8 to 12 cycles; 72 o C 5min; 10 o C hold) Purify the PCR product with MinElute column: a) Pool all of PCR reactions into 1.5ml Lowbind tube. b) Place the tube of beads in a magnetic rack and transfer the supernatant to a fresh tube. c) Perform the clean up following the manufacturer s manual. d) Elute the DNA with 30ul EB.
21 9. Gel purify the final library and QC Reagents: NuSieve 3:1 Agarose from Lonza Qiaquick gel purificaiton kit from Qiagen TrackIt 25bp ladder from Invitrogen SYBR Gold nucleic acid gel stain from Invitrogen a) Prepare a 2% agarose gel in 1XTAE buffer b) Load 25bp ladder to the left well, skip a well, and load all materials from last step evenly among one or multiple wells. c) Run the gel at 90V until the yellow dye in the marker is close to the bottom of the gel. d) Stain the gel with SYBR Gold for 30mins. e) Exercise the ~200 bp band with a clean razor blade. f) Process gel purification following the manufacturer s manual. g) QC the final library molecules with Pico/Bioanalyzer/qPCR.
NimbleGen 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 informationThe RNAi Consortium (TRC) Broad Institute
TRC Laboratory Protocols Protocol Title: One Step PCR Preparation of Samples for Illumina Sequencing Current Revision Date: 11/10/2012 RNAi Platform,, trc_info@broadinstitute.org Brief Description: This
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 informationWhole genome Bisulfite Sequencing for Methylation Analysis Preparing Samples for the Illumina Sequencing Platform
Whole genome Bisulfite Sequencing for Methylation Analysis Preparing Samples for the Illumina Sequencing Platform Introduction, 2 Sample Prep Workflow, 3 Best Practices, 4 DNA Input Recommendations, 6
More informationReduced Representation Bisulfite Sequencing for Methylation Analysis Preparing Samples for the Illumina Sequencing Platform
Reduced Representation Bisulfite Sequencing for Methylation Analysis Preparing Samples for the Illumina Sequencing Platform Introduction, 3 Sample Prep Workflow, 4 Best Practices, 5 DNA Input Recommendations,
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 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 information1) Vector Preparation sg 1371 w/blp1 Ef1α puro t29 BFP (602 ng/ul)
1) Vector Preparation sg 1371 w/blp1 Ef1α puro t29 BFP (602 ng/ul) a) Digest 5 ug of vector with Thermo Scientific FastDigest BstX1 and Blp1 for 1h at 37ºC. Set up reaction as follows: 100 ul Reaction
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 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 informationAmplicon Template Preparation and Sequencing
Please note: the shared 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 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 informationPCR 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 informationWelcome to Pacific Biosciences' Introduction to SMRTbell Template Preparation.
Introduction to SMRTbell Template Preparation 100 338 500 01 1. SMRTbell Template Preparation 1.1 Introduction to SMRTbell Template Preparation Welcome to Pacific Biosciences' Introduction to SMRTbell
More informationPaired-End Sample Preparation Guide
Paired-End Sample Preparation Guide FOR RESEARCH USE ONLY Introduction 3 Sample Prep Workflow 4 Best Practices 5 DNA Input Recommendations 7 Kit Contents 9 Consumables and Equipment 11 Fragment DNA 13
More informationUpdated: July 2005. 5' End label RNA markers 18.113 (18mer) and 44.12 (24mer) with Kinase and 32P-gamma-ATP. Gel purify labeled markers.
RNA Cloning Method Flowchart Extract total RNA containing small RNAs. Check quality on denaturing gel with EtBr staining 5' End label RNA markers 18.113 (18mer) and 44.12 (24mer) with Kinase and 32P-gamma-ATP.
More informationMate Pair Library v2 Sample Preparation Guide
Mate Pair Library v2 Sample Preparation Guide For 2 5 kb Libraries For Research Use Only Not for use in diagnostic procedures Topics 3 Introduction 6 Best Practices 9 Data Analysis and Interpretation 11
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 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 informationPaired-End Sequencing Sample Preparation Guide
Paired-End Sequencing Sample Preparation Guide FOR RESEARCH USE ONLY Topics 3 Introduction 5 Best Practices 6 DNA Input Recommendations 7 Paired-End Sample Preparation Kit Contents 9 User-Supplied Consumables
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 informationHighPure Maxi Plasmid Kit
HighPure Maxi Plasmid Kit For purification of high pure plasmid DNA with high yields www.tiangen.com PP120109 HighPure Maxi Plasmid Kit Kit Contents Storage Cat.no. DP116 Contents RNaseA (100 mg/ml) Buffer
More informationHow To Write An Ipa
LIBRARY PREPARATION NEBNext Multiplex Small RNA Library Prep Set for Illumina (Set 1) Instruction Manual NEB #E7300S/L 24/96 reactions Sign up for the NEBNext e-newsletter Scan this code or visit www.neb.com/
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 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 informationChromatin Immunoprecipitation
Chromatin Immunoprecipitation A) Prepare a yeast culture (see the Galactose Induction Protocol for details). 1) Start a small culture (e.g. 2 ml) in YEPD or selective media from a single colony. 2) Spin
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 informationUSER GUIDE. Encore PART NOS. 8041 and 8042. SP Rapid Library Systems
USER GUIDE Encore PART NOS. 8041 and 8042 SP Rapid Library Systems Patents, Licensing and Trademarks 2012 2013 NuGEN Technologies, Inc. All rights reserved. The Encore, Ovation and Applause families of
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 informationInverse PCR & Cycle Sequencing of P Element Insertions for STS Generation
BDGP Resources Inverse PCR & Cycle Sequencing of P Element Insertions for STS Generation For recovery of sequences flanking PZ, PlacW and PEP elements E. Jay Rehm Berkeley Drosophila Genome Project I.
More informationReal-time quantitative RT -PCR (Taqman)
Real-time quantitative RT -PCR (Taqman) Author: SC, Patti Lab, 3/03 This is performed as a 2-step reaction: 1. cdna synthesis from DNase 1-treated total RNA 2. PCR 1. cdna synthesis (Advantage RT-for-PCR
More informationChromatin Immunoprecipitation (ChIP)
Chromatin Immunoprecipitation (ChIP) Day 1 A) DNA shearing 1. Samples Dissect tissue (One Mouse OBs) of interest and transfer to an eppendorf containing 0.5 ml of dissecting media (on ice) or PBS but without
More informationSOLIDscript Solid Phase cdna Synthesis Kit Instruction Manual
Toll Free: 866-252-7771 752A Lincoln Blvd. Phone: 732-469-7771 Fax: 732-469-7782 Middlesex, NJ 08846 Web: www.purebiotechllc.com SOLIDscript Solid Phase cdna Synthesis Kit Instruction Manual Product: SOLIDscript
More informationTransformAid Bacterial Transformation Kit
Home Contacts Order Catalog Support Search Alphabetical Index Numerical Index Restriction Endonucleases Modifying Enzymes PCR Kits Markers Nucleic Acids Nucleotides & Oligonucleotides Media Transfection
More informationSequencing Library qpcr Quantification Guide
Sequencing Library qpcr Quantification Guide FOR RESEARCH USE ONLY Introduction 3 Quantification Workflow 4 Best Practices 5 Consumables and Equipment 6 Select Control Template 8 Dilute qpcr Control Template
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 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 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 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 informationContents. XI. Materials and Equipment Needed But Not Provided 5. DNA Extraction from Small Amount of Tissue 10
Contents I. Overview 1 II. Kit Components 1 III. Storage 2 IV. Intended Use 2 V. Safety Warnings and Precautions 2 VI. Warranty and Liability 2 VII. Technical Assistance 3 VIII. Quality Management 3 IX.
More informationRT-PCR: Two-Step Protocol
RT-PCR: Two-Step Protocol We will provide both one-step and two-step protocols for RT-PCR. We recommend the twostep protocol for this class. In the one-step protocol, the components of RT and PCR are mixed
More informationNimbleGen SeqCap EZ Library SR User s Guide Version 3.0
NimbleGen SeqCap EZ Library SR User s Guide Version 3.0 For life science research only. Not for use in diagnostic procedures. Copyright 2011 Roche NimbleGen, Inc. All Rights Reserved. Editions Version
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 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 informationABSTRACT. Promega Corporation, Updated September 2008. http://www.promega.com/pubhub. 1 Campbell-Staton, S.
A Modified Wizard SV Genomic DNA Purification System Protocol to Purify Genomic DNA... A Modified Wizard SV Genomic DNA Purification System Protocol to Purify Genomic DNA from Shed Reptile Skin ABSTRACT
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 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 informationBioanalyzer Applications for
Bioanalyzer Applications for Next-Gen Sequencing: Updates and Tips March 1 st, 2011 Charmian Cher, Ph.D Field Applications Scientist Page 1 Agenda 1 2 3 Next-gen sequencing library preparation workflow
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 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 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 informationImproved methods for site-directed mutagenesis using Gibson Assembly TM Master Mix
CLONING & MAPPING DNA CLONING DNA AMPLIFICATION & PCR EPIGENETICS RNA ANALYSIS Improved methods for site-directed mutagenesis using Gibson Assembly TM Master Mix LIBRARY PREP FOR NET GEN SEQUENCING PROTEIN
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 informationSOP Title: Multiplex-PCR check of genomic DNA isolated from FFPE tissue for its usability in array CGH analysis
SOP Title: Multiplex-PCR check of genomic DNA isolated from FFPE tissue for its usability in array CGH analysis The STORE processing methods were shown to be fit-for purpose for DNA, RNA and protein extraction
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 informationKevin Bogart and Justen Andrews. Extraction of Total RNA from Drosophila. CGB Technical Report 2006-10 doi:10.2506/cgbtr-200610
Kevin Bogart and Justen Andrews Extraction of Total RNA from Drosophila CGB Technical Report 2006-10 doi:10.2506/cgbtr-200610 Bogart K and Andrews J. 2006. Extraction of Total RNA from Drosophila. CGB
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 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 informationRecombinant DNA & Genetic Engineering. Tools for Genetic Manipulation
Recombinant DNA & Genetic Engineering g Genetic Manipulation: Tools Kathleen Hill Associate Professor Department of Biology The University of Western Ontario Tools for Genetic Manipulation DNA, RNA, cdna
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 informationncounter Gene Expression Assay Manual Total RNA and Cell Lysate Protocols
ncounter Gene Expression Assay Manual Total RNA and Cell Lysate Protocols v.20090807 For research use only. Not for use in diagnostic procedures. Limited License Subject to the terms and conditions of
More informationRNA Antisense Purification (RAP): Experimental Protocols
RNA Antisense Purification (RAP): Experimental Protocols Jesse Engreitz engreitz@broadinstitute.org August 23, 2014. Last Updated: October 18, 2014 This document describes the experimental procedures for
More informationDNA Isolation Kit for Cells and Tissues
DNA Isolation Kit for Cells and Tissues for 10 isolations of 00 mg each for tissue or 5 x 10 7 cultured cells Cat. No. 11 81 770 001 Principle Starting material Application Time required Results Benefits
More informationInvestigating a Eukaryotic Genome: Cloning and Sequencing a Fragment of Yeast DNA
Investigating a Eukaryotic Genome: Cloning and Sequencing a Fragment of Yeast DNA Credits: This lab was created by Sarah C.R. Elgin and developed and written by Kathleen Weston-Hafer. Specific protocols
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 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 informationAffymetrix Tiling Arrays
Affymetrix Tiling Arrays Updated: S. Silverman 6/2011 Updated by Mark Hickman (Nov 2009). Updated by David Gresham and Alex Ward (June 2008). This protocol is based on previous protocols developed by Maitreya
More informationMolecular Cloning, Product Brochure
, Product Brochure Interest in any of the products, request or order them at Bio-Connect. Bio-Connect B.V. T NL +31 (0)26 326 44 50 T BE +32 (0)2 503 03 48 Begonialaan 3a F NL +31 (0)26 326 44 51 F BE
More informationQUANTITATIVE RT-PCR. A = B (1+e) n. A=amplified products, B=input templates, n=cycle number, and e=amplification efficiency.
QUANTITATIVE RT-PCR Application: Quantitative RT-PCR is used to quantify mrna in both relative and absolute terms. It can be applied for the quantification of mrna expressed from endogenous genes, and
More informationqpcr Quantification Protocol Guide
qpcr Quantification Protocol Guide FOR RESEARCH USE ONLY Topics 3 Introduction 5 User-Supplied Consumables and Equipment 7 Select Template 8 Dilute qpcr Template 9 Dilute Libraries 10 Prepare Reaction
More informationSection III: Loading and Running DNA in Agarose Gels
Section III: In This Section DNA Loading 90 Loading Buffers 91 Optimal Voltage and Electrophoretic Times 92 Fast Running Protocols for High Resolution in MetaPhor Agarose Gels 93 References 94 89 Section
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 informationRAGE. Plugs for RAGE/PFGE
1 RAGE Rotating Field Gel Electrophoresis (RAGE) is a variation on Pulsed Field Gel Electrophoresis (PFGE) and gives similar results. We use equipment that was only briefly marketed by Stratagene, at far
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 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 informationAgencourt RNAdvance Blood Kit for Free Circulating DNA and mirna/rna Isolation from 200-300μL of Plasma and Serum
SUPPLEMENTAL PROTOCOL WHITE PAPER Agencourt RNAdvance Blood Kit for Free Circulating DNA and mirna/rna Isolation from 200-300μL of Plasma and Serum Bee Na Lee, Ph.D., Beckman Coulter Life Sciences Process
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 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 informationPrimeSTAR HS DNA Polymerase
Cat. # R010A For Research Use PrimeSTAR HS DNA Polymerase Product Manual Table of Contents I. Description...3 II. III. IV. Components...3 Storage...3 Features...3 V. General Composition of PCR Reaction
More informationHow To Shear Chromatin
truchip Low Cell Chromatin Shearing Kit with Non-ionic Shearing Buffer Part Number: 010144 Rev C 1 Page INTRODUCTION The truchip Low Cell Chromatin Shearing Kit with Non-ionic Shearing Buffer (PN 520084)
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 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 informationPCR was carried out in a reaction volume of 20 µl using the ABI AmpliTaq GOLD kit (ABI,
Supplemental Text/Tables PCR Amplification and Sequencing PCR was carried out in a reaction volume of 20 µl using the ABI AmpliTaq GOLD kit (ABI, Foster City, CA). Each PCR reaction contained 20 ng genomic
More informationCluster Generation. Module 2: Overview
Cluster Generation Module 2: Overview Sequencing Workflow Sample Preparation Cluster Generation Sequencing Data Analysis 2 Cluster Generation 3 5 DNA (0.1-5.0 μg) Library preparation Single Cluster molecule
More informationTruSeq DNA Methylation Library Preparation Guide
TruSeq DNA Methylation Library Preparation Guide Kit Contents 3 Consumables and Equipment 4 Preparation 5 Quality Control of Bisulfite-Converted DNA 6 TruSeq DNA Methylation Kit Protocol 7 Sequencing the
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 informationCompleteⅡ 1st strand cdna Synthesis Kit
Instruction Manual CompleteⅡ 1st strand cdna Synthesis Kit Catalog # GM30401, GM30402 Green Mountain Biosystems. LLC Web: www.greenmountainbio.com Tel: 800-942-1160 Sales: Sales@ greenmountainbio.com Support:
More informationTransformation Protocol
To make Glycerol Stocks of Plasmids ** To be done in the hood and use RNase/DNase free tips** 1. In a 10 ml sterile tube add 3 ml autoclaved LB broth and 1.5 ul antibiotic (@ 100 ug/ul) or 3 ul antibiotic
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 informationDetailed protocol: Combined method for RNA isolation. from cartilage
Detailed protocol: Combined method for RNA isolation from cartilage REAGENTS - chloroform - DNase (RNase-free DNase Set, cat.no. 79254, Qiagen, Hilden, Germany) - 80 % Ethanol (in DEPC-treated water) -
More informationMystiCq microrna cdna Synthesis Mix Catalog Number MIRRT Storage Temperature 20 C
microrna cdna Synthesis Mix Catalog Number MIRRT Storage Temperature 20 C Product Description The microrna cdna Synthesis Mix has been designed to easily convert micrornas into cdna templates for qpcr
More informationPicoMaxx High Fidelity PCR System
PicoMaxx High Fidelity PCR System Instruction Manual Catalog #600420 (100 U), #600422 (500 U), and #600424 (1000 U) Revision C Research Use Only. Not for Use in Diagnostic Procedures. 600420-12 LIMITED
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 informationTaq98 Hot Start 2X Master Mix
Taq98 Hot Start 2X Master Mix Optimized for 98C Denaturation Lucigen Corporation 2905 Parmenter St, Middleton, WI 53562 USA Toll Free: (888) 575-9695 (608) 831-9011 FAX: (608) 831-9012 lucigen@lucigen.com
More informationUSER GUIDE. qpcr System. Ovation. (formerly WT-Ovation RNA Amplification System) PART NO. 2210
USER GUIDE Ovation qpcr System (formerly WT-Ovation RNA Amplification System) PART NO. 2210 Patents, Licensing and Trademarks 2009 2013 NuGEN Technologies, Inc. All rights reserved. The Encore, Ovation
More informationAutomation in Genomics High-throughput purification of nucleic acids from biological samples. Valentina Gualdi Operational Scientist PGP
Automation in Genomics High-throughput purification of nucleic acids from biological samples Valentina Gualdi Operational Scientist PGP OVERVIEW Nucleic acid purification technologies general aspects Genomic
More informationGlobal MicroRNA Amplification Kit
Global MicroRNA Amplification Kit Store kit at -20 C on receipt (ver. 3-060901) A limited-use label license covers this product. By use of this product, you accept the terms and conditions outlined in
More informationqstar mirna qpcr Detection System
qstar mirna qpcr Detection System Table of Contents Table of Contents...1 Package Contents and Storage Conditions...2 For mirna cdna synthesis kit...2 For qstar mirna primer pairs...2 For qstar mirna qpcr
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 informationCLONING IN ESCHERICHIA COLI
CLONING IN ESCHERICHIA COLI Introduction: In this laboratory, you will carry out a simple cloning experiment in E. coli. Specifically, you will first create a recombinant DNA molecule by carrying out a
More information