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High-Sensitive DNA Library Preparation Kit (Illumina)

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1. EpiNext High Sensitive DNA Library Preparation Kit Illumina Base Catalog P 1053 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiNext High Sensitive DNA Library Preparation Kit Illumina is suitable for preparing a DNA library using sub nanogram amounts of DNA input for next generation sequencing applications using an Illumina sequencer These applications include genomic DNA seq ChIP seq MeDIP hMeDIP seg classical bisulfite seg and targeted re sequencing The optimized protocol and components of the kit allow both non barcoded singleplexed and barcoded multiplexed DNA libraries to be constructed quickly with reduced bias Starting Material and Input Amount Starting materials can include fragmented dsDNA isolated from various tissue or cell samples dsDNA enriched from a ChIP reaction MeDIP hMeDIP reaction or exon capture DNA should be relatively free of RNA because large fractions of RNA will impair end repair and dA tailing resulting in reduced ligation capabilities The input amount of DNA can be from 0 2 ng to 100 ng For optimal preparation the input amount should be 10 ng to 50 ng Precautions To avoid cross contamination carefully pipette the sample or solution into the tube vials Use aerosol barrier pipette tips and always change pipette tips between liquid transfers Wear gloves throughout the entire procedure In case of contact between gloves and sample change gloves immediately 110 Bi County Blv
2. The end repair dA tailing end polishing of the DNA fragments are performed simultaneously Adaptors are then ligated to both ends of the polished DNA fragments for amplification and sequencing Ligated fragments are size selected and purified with MQ beads which allows quick and precise size selection of DNA Size selected DNA fragments are amplified with high fidelity PCR Mix that ensures maximum yields from minimum amounts of starting material and provides highly accurate amplification of library DNA with low error rates and minimum bias DNA End Polishing Adaptor Ligation Size Selection Optional Amplification NGS Illumina Fig 1 Workflow of the EpiNext High Sensitive DNA Library Preparation Kit 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 4 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1053 Fl Sample 11 60 z s0 40 30 20 10 a0 300 200 8600 1500 10380 bp Fig2 Size distribution of library fragments Human placenta DNA was sheared to around 300 bps in peak size and 0 2 ng of DNA was used for DNA library preparation using EpiNext High Sensitive DNA Library Preparation Kit Illumina ASSAY PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Fragmented
3. 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P1055 Repeat Step 3 2e two times for total of three washes Open the PCR tube cap and air dry beads for 10 minutes while the tube is on the magnetic stand Resuspend the beads in 12 ul Elution Buffer and incubate at room temperature for 2 minutes to release the DNA from the beads Capture the beads by placing the tube in the magnetic stand for 4 minutes or until the solution is completely clear Transfer 11 ul to a new 0 2 ml PCR tube for PCR amplification 4 Library Amplification a Prepare the PCR Reactions Thaw all reaction components including master mix DNA RNA free water primer solution and DNA template Mix well by vortexing briefly Keep components on ice while in use and return to 20 C immediately following use Add components into each PCR tube well according to the following table Component Size ul HiFi Master Mix 2X 12 5 ul Primer I or barcode Adaptor Ligated DNA 10 5 ul Total Volume 25W Important Note Use of Primer I included in the kit will generate a singleplexed library For multiplexed library preparation replace Primer I with one of the12 different barcodes indexes contained in the EpiNext NGS Barcode Index Set 12 Cat No P 1060 You can also add user defined barcodes Illumina compatible instead
4. ChIP reactions and dSDNA enriched from MeDIP hMeDIP reactions or exon capture e Fast and streamlined procedure the procedure from fragmented DNA to size selection is less than 1h 30 min No clean up is required between each step and all reactions take place in the same tube thereby saving time and preventing handling errors as well as loss of valuable samples Gel free size selection further reduces the preparation time 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 3 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P1055 e The most convenient for use the kit contains all required components for each step of DNA library preparation which are sufficient for end polishing ligation clean up size selection and library amplification thereby allowing the library preparation to be the most convenient with reliable and consistent results e Minimized bias Ultra HiFi amplification enables to reproducibly achieve high yields of DNA library with minimal sequence bias and low error rates PRINCIPLE amp PROCEDURE The EpiNext High Sensitive DNA Library Preparation Kit Illumina contains all reagents required at each step of workflow for carrying out successful DNA library preparation In the library preparation DNA is first fragmented to appropriate size about 300 bps in peak size
5. contain DNA g Keep the PCR tube in the magnetic stand and add 200 ul of freshly prepared 80 ethanol to the tube Incubate at room temperature for 1 min and then carefully remove and discard the ethanol h Repeat Step 3 1g one time for total of two washes i Open the PCR tube cap and air dry beads for 10 minutes while the tube is on the magnetic stand j Resuspend the beads in 12 ul Elution Buffer and incubate at room temperature for 2 minutes to release the DNA from the beads k Capture the beads by placing the tube in the magnetic stand for 4 minutes or until the solution is completely clear l Transfer 11 ul to a new 0 2 ml PCR tube for PCR amplification 3 2 Clean up of Ligated DNA Optional a Resuspend MQ Binding Beads by vortex b Add 34 ul of resuspended beads to the PCR tube of ligation reaction Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times C Incubate for 5 minutes at room temperature to allow DNA to bind to beads d Put the PCR tube on an appropriate magnetic stand until the solution is clear about 4 minutes Carefully remove and discard the supernatant Be careful not to disturb or discard the beads that contain DNA e Keep the PCR tube in the magnetic stand and add 200 ul of freshly prepared 80 ethanol to the tube Incubate at room temperature for 1 min and then carefully remove and discard the ethanol 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 7 Tel 1 877
6. is properly processed with proper amount of input DNA Over amplification of library PCR artifacts from over amplification of the library may cause the fragment population to shift higher than expected Make sure to use proper PCR cycles to avoid this problem RELATED PRODUCTS DNA Isolation and Cleanup P 1003 FitAmp General Tissue Section DNA Isolation Kit P 1004 FitAmp Plasma Serum DNA Isolation Kit P 1006 DNA Concentrator Kit P 1007 FitAmp Gel DNA Isolation Kit P 1009 FitAmp Paraffin Tissue Section DNA Isolation Kit P 1017 FitAmp Urine DNA Isolation Kit P 1018 FitAmp Blood and Cultured Cell DNA Extraction Kit Sonication Instruments EQC 1100 EpiSonic Multi Functional Bioprocessor 1100 DNA Enrichment Reaction P 1015 Methylamp Methylated DNA Capture Kit P 1038 EpiQuik Hydroxymethylated DNA Immunoprecipitation nMeDIP Kit P 1052 EpiQuik MeDIP Ultra Kit P 2002 EpiQuik Chromatin Immunoprecipitation Kit P 2003 EpiQuik Tissue Chromatin Immunoprecipitation Tissue Kit P 2014 EpiQuik Plant ChIP Kit P 2025 ChromaFlash One Step ChIP Kit P 2026 ChromaFlash One Step Magnetic ChIP kit P 2027 ChromaFlash ChIP Ultra Kit DNA Bisulfite Conversion P 1001 Methylamp DNA Modification KIt P 1026 BisulFlash DNA Modification Kit PCR Analysis P 1028 Methylamp MS qPCR Fast Kit P 1029 EpiQuik Quantitative PCR Fast Kit 110 Bi County Blvd Ste 122 Farmingdale NY 1173
7. 5 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 10 Printed 2013 12 31 P1053 DNA Library Prep P 1051 EpiNext DNA Library Preparation Kit P 1055 EpiNext Post Bisulfite DNA Library Preparation Kit Illumina NGS Barcode P 1060 EpiNext NGS Barcode Index Set 12 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 11 Printed 2013 12 31 P 1053
8. best results the amount DNA of input DNA should be gt 10 ng Insufficient purity of starting DNA Ensure that RNA is removed by Rnase treatment before starting library preparation protocol Improper reaction conditions at Check if the reagents are properly each reaction step added and incubation temperature and time are correct at each reaction step including End Polishing Adaptor Ligation size Selection and Amplification Improper storage of the kit Ensure that the kit has not exceeded the expiration date Standard shelf life when stored properly is 6 months from date of receipt Unexpected peak size Improper ratio of MQ Binding Check if the correct volume of MQ 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 9 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P1055 of Agilent bioanalyzer Beads to DNA volume in size trace Presence of selection lt 150 bp adaptor dimers or presence of Binding Beads is added to the DNA solution accordingly Proper ratios should remove the fragments of unexpected peak size larger fragments than expected Insufficient ligation Too much and too little input DNA may cause insufficient ligation which can shift the peak size of the fragment population to be shorter or larger than expected Make sure that the ligation reaction
9. d Ste 122 Farmingdale NY 11735 Page 1 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1053 KIT CONTENTS Component 12 reactions 24 reactions Storage Cat P 1053 12 Cat P 1053 24 Upon Receipt MQ Binding Beads 2X HiFi PCR Master Mix 160 ul 320 ul Primer I 10 uM C C C C C C C C C Elution Buffer 1000 ul 2000 ul pe Primer U 10 uM 15 pl 30 yl T User Guide RT Spin the solution down to the bottom prior to use SHIPPING amp STORAGE The kit is shipped on frozen ice packs at 4 C Upon receipt Store the following components at 20 C immediately 10X End Polishing Buffer End Polishing Enzyme Mix End Polishing Enhancer 2X Ligation Buffer T4 DNA Ligase Adaptors 2X HiFi PCR Master Mix Primer U Primer I and Elution Buffer Store the following components at 4 C MQ Binding Beads Store all other components at room temperature MATERIALS REQUIRED BUT NOT SUPPLIED O Vortex mixer Sonicator or enzymes for DNA fragmentation Agilent Bioanalyzer or comparable method to assess the quality of DNA library Thermocycler Centrifuge including desktop centrifuge up to 14 000 rpm E E E E O Magnetic stand 96 well format O Pipettes and pipette tips O PCR tubes or plates O 1 5 ml microcentrifuge tubes E 80 Ethanol 110 Bi County B
10. dsDNA that is isolated from various tissues or cell samples 0 2 ng 100 ng optimized 10 50 ng per preparation dsDNA enriched from a ChIP reaction MeDIP hMeDIP reaction or exon capture 0 2 ng 100 ng DNA should be of high quality and relatively free of RNA RNAse can be used to remove RNA and DNA should be eluted in DNase RNase free water DNA Fragmentation dsDNA enriched from a ChIP reaction MeDIP hMeDIP reaction or exon capture should already be fragmented DNA isolated from various tissue or cell samples can be fragmented using one of the following methods For the best results we highly recommend using a waterbath based sonication 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 5 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1053 device The peak size of fragmented DNA should be compatible with the read length of the Illumina sequencing platform to be used In general the peak size of fragments should be 200 300 bps Waterbath Sonication Epigentek s EpiSonic 1100 Epigentek Cat No EQC 1100 For target peak size 200 bps use 10 ul of DNA solution standard amount 10 50 ng per 0 2 ml PCR tube Shear 40 cycles under cooling condition 45 seconds On 15 seconds Off each at 110 120 watts For more detailed information of use please see the DNA Shearing Protocol f
11. e ethanol Repeat Step 7e two times for a total of three washes Open the PCR tube cap and air dry beads for 10 minutes while the tube is on the magnetic stand Resuspend the beads in 22 ul Elution Buffer and incubate at room temperature for 2 minutes to release the DNA from the beads Capture the beads by placing the tube in the magnetic stand for 4 minutes or until the solution is completely clear Transfer 20 ul to a new 0 2 ml PCR tube Quality of the prepared library can be assessed using an Agilent Bioanalyzer or comparable method Library fragments should have the correct size distribution ex 300 bps at peak size without adaptors or adaptor dimers To check the size distribution dilute library 5 fold with water and apply it to an Agilent high sensitivity chip If there is presence of lt 150 bp adaptor dimers or of larger fragments than expected they should be removed To remove fragments below 150 bps use 0 8X MQ Binding Beads ex add 16 ul of MQ Binding Beads to 20 ul of sample according to steps a i of section 5 Clean up of Amplified Library DNA To remove fragments above 500 bps follow steps a l of section 3 2 Size Selection of Ligated DNA The prepared DNA library can be quantified with various DNA library quantification methods The prepared library DNA can be stored at 20 C until ready to use for sequencing TROUBLESHOOTING Possible Cause Suggestion Low yield of library Insufficient amount of starting To obtain the
12. ed singleplexed and barcoded multiplexed DNA library preparation and are fully compatible with Illumina platforms such as MiSeq or HiSeq sequencers 2 If using adaptors from other suppliers both single end and barcode adaptors make sure they are compatible with Illumina platforms and add the correct amount final concentration 1 5 2 uM or according to the supplier s instruction 3 Size Selection Clean up 3 1 Size selection of Ligated DNA Note If the starting DNA amount is less than 50 ng size selection is not recommended and alternatively clean up of ligated DNA can be performed prior to PCR amplification according to step 3 2 of the protocol a Resuspend MQ Binding Beads by vortex b Add 14 ul of resuspended MQ Binding Beads to the tube of ligation reaction Mix well by pipetting up and down at least 10 times C Incubate for 5 minutes at room temperature d Put the tube on an appropriate magnetic stand until the solution is clear about 4 minutes Carefully transfer the supernatant containing DNA to a new tube Caution do not discard the supernatant Discard the beads that contain the unwanted large fragments e Add 10 ul resuspended beads to the supernatant mix well and incubate for 5 minutes at room temperature f Put the PCR tube on an appropriate magnetic stand until the solution is clear about 4 minutes Carefully remove and discard the supernatant Be careful not to disturb or discard the beads that
13. he EpiNext High Sensitive DNA Library Preparation Kit Illumina and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW DNA library preparation is a critical step for next generation sequencing NGS For generating accurate sequencing data in NGS the prepared library DNA should be sufficient in yield and of high quality Also as NGS technology is continuously improving DNA library preparation is required to be optimized accordingly For example most of the currently used methods are time consuming expensive inconvenient and specifically need large amounts of DNA These reactions result ina DNA library preparation which cannot be used for biological samples with limited amounts of starting material such as tumor biopsy early embryos embryonic tissues and circulating DNA In addition the amount of DNA enriched by ChIP or MeDIP hmeDIP is often at low or sub nanogram levels which causes insufficient DNA library yields To address this issue Epigentek offers the EpiNext High Sensitive DNA Library Preparation Kit Illumina This kit has the following features e High sensitivity and flexibility Can be used for both non barcoded singleplexed and barcoded multiplexed DNA library preparation The amount of input DNA can be as low as 0 2 ng with a range from 0 2 to 100 ng Various dsDNA can be used which includes limited amounts of fragmented dsDNA isolated from various tissue or cell samples dsDNA enriched from
14. lvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 2 Printed 2013 12 31 P 1053 O Distilled water O DNA sample GENERAL PRODUCT INFORMATION Quality Control Each lot of EpiNext High Sensitive DNA Library Preparation Kit Illumina is tested against predetermined specifications to ensure consistent product quality Epigentek guarantees the performance of all products in the manner described in our product instructions Product Warranty If this product does not meet your expectations simply contact our technical support unit or your regional distributor We also encourage you to contact us if you have any suggestions about product performance or new applications and techniques Safety Suitable lab coat disposable gloves and proper eye protection are required when working with this product Product Updates Epigentek reserves the right to change or modify any product to enhance its performance and design The information in this User Guide is subject to change at any time without notice Thus only use the User Guide that was supplied with the kit when using that kit Usage Limitation The EpiNext High Sensitive DNA Library Preparation Kit Illumina is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property T
15. of Primer I Program the PCR Reactions Place the reaction plate in the instrument and set the PCR conditions as follow Cycle Step Time Cycle Activation Boses t 98 se 20 sec Cycling 55 C 20 sec Variable 72 C 20 sec PCR cycles may vary depending on the inout DNA amount In general use 10 PCR cycles for 100 ng 12 cycles for 50 ng 16 cycles for 5 ng 18 cycles for 1 ng and 22 cycles for 0 2 ng DNA input Further optimization of PCR cycle number may be required 5 Clean up of Amplified Library DNA a Resuspend MQ Binding Beads by vortex 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 8 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1053 j Add 25 ul of resuspended beads to the PCR tube of amplification reaction Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times Incubate for 5 minutes at room temperature to allow DNA to bind to beads Put the PCR tube on an appropriate magnetic stand until the solution is clear about 4 minutes Carefully remove and discard the supernatant Be careful not to disturb or discard the beads that contain DNA Keep the PCR tube in the magnetic stand and add 200 ul of freshly prepared 80 ethanol to the tube Incubate at room temperature for 1 min and then carefully remove and discard th
16. or EpiSonic 1100 If using other waterbath sonicators please follow the supplier s instruction Enzymatic Shearing The DNA can also be sheared using various enzyme based methods Optimization of the shearing conditions for example enzyme concentration and incubation time is needed in order to use enzyme based methods 1 DNA End Polishing Prepare end repair reaction in a 0 2 ml PCR tube according to Table 1 Table 1 End Polishing Reaction Fragmented DNA 10 ul 10 50 ng 10X End Polishing Buffer 1 5 ul Toivomme peo Mix and incubate for 20 min at 25 C and 20 min at 72 C in a thermocycler without heated lid Note the amount of fragmented DNA can be 0 2 100 ng with an optimal amount of 10 50 ng 2 Adaptor Ligation Prepare a reaction mix for adaptor ligation according to Table 1 Add the following reagents to a 0 2 ml PCR tube containing end polished DNA from step 1 Table 2 Adaptor Ligation End polished DNA from step 1 15 ul 2X Ligation Buffer T4 DNA Ligase Total Volume a 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 6 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1053 b Mix and incubate for 15 min at 25 C in a thermocycler without heated lid Note 1 The pre annealed adapters included in the kit are suitable for both non barcod

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