EpiNext™ DNA Size Selection Kit
Contents
1. EpiNext DNA Size Selection Kit Base Catalog P 1059 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiNext DNA Size Selection Kit utilizes magnetic bead technology for quick DNA size selection in a high throughput format The kit is suitable for removing DNA fragments of lt 150 bps for DNA library preparation for Illumina Life Technologies SOLID lon Torrent and Roche 454 next generation sequencing applications It can also be used for removing undesired larger DNA fragments by optimizing the bead to DNA volume ratio accordingly The indicated number of reactions can be performed for a standard 50 ul solution input DNA sample Starting Material and Input amount DNA fragments of various lengths Input amount can be from 0 1 ng to 1 ug 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 Blvd 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 2014 02 27 Epigentek Group Inc All rights reserved Products are for research use only P 1059 KIT CONTENTS Component 48 reactions 96 reactions Storage Cat P 1059 48 Cat P 1059 96 Upon Rec2. 7 ChromaFlash High Sensitivity ChIP Kit 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 7 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 02 27 Epigentek Group Inc All rights reserved Products are for research use only P 1059 PCR Analysis P 1029 EpiQuik Quantitative PCR Fast Kit DNA Library Prep P 1051 EpiNext DNA Library Preparation Kit Illumina P 1053 EpiNext High Sensitivity DNA Library Preparation Kit Illumina P 1055 EpiNext Post Bisulfite DNA Library Preparation Kit Illumina 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 2014 02 27 Epigentek Group Inc All rights reserved Products are for research use only P 1059
3. e 2 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 Printed 2014 02 27 P 1059 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 DNA Size Selection Kit is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The EpiNext DNA Size Selection Kit and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW DNA size selection is necessary after DNA shearing as part of the library construction process for next generation sequencing regardless of the platform used Obtaining high recovery of selected DNA fragments is critical for the reduction of sequencing bias The EpiNext DNA Size Selection Kit is optimized for DNA fragment size selection used for various next generation sequencing platforms including Illumina Life Technologies SOLID lon Torrent and Roche 454 The EpiNext DNA Size Selection Kit has the following features e Optimized fragment selection chemistries for complete separation of DNA fragments according to size Suitable for Illumina SOLID lon Torrent a
4. e selection of DNA fragments Human Placenta DNA was sheared to 100 700 bps in length with peak size of about 300 bps Fig A A target peak size of 500 bps Fig B was selected using the EpiNext DNA Size Selection 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 2014 02 27 Epigentek Group Inc All rights reserved Products are for research use only P 1059 ASSAY PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Fragmented dsDNA isolated from various tissues or cell samples 0 2 ng 500 ng optimized 20 100 ng per preparation dsDNA enriched from a ChIP reaction MeDIP hMeDIP reaction or exon capture 0 2 ng 100 ng cDNA or dsDNA converted from reverse transcription of RNA or bisulfite treatment of DNA DNA should be high quality and relatively free of RNA RNAse can be used to remove RNA and DNA should be eluted in DNase RNase free water For the magnetic stand used to capture DNA bound MQ beads we recommend using Epigentek s EpiMag HT Magnetic Separator which is very strong and proven to quickly and efficiently achieve high reproducible retention of magnetic bead bound DNA in a single PCR tube and in various 96 well plates Size Selection of DNA Fragments Note If the starting amount of fragmented DNA is less than 50 ng or w
5. eipt MQ Binding Beads 4 ml 8 ml 4 C Elution Solution 1ml 2ml 4 C 96 well plate 1 fl RT User Guide 1 1 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 4 C MQ Binding Beads Elution Solution Store all other components at room temperature The kit is stable for at least 6 months from the shipment date when stored properly MATERIALS REQUIRED BUT NOT SUPPLIED O Vortex mixer Agilent Bioanalyzer or comparable method to assess the quality of the DNA library 0 2 ml PCR tubes 80 ethanol DNA sample 0 OF O 0 O Magnetic stand 96 well format O Pipettes and pipette tips GENERAL PRODUCT INFORMATION Quality Control Each lot of EpiNext DNA Size Selection Kit 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 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Pag
6. hen large size DNA fragments are not a concern Protocol I can be performed to remove DNA fragments lt 150 bps e g primers primer dimers adaptors and adaptor dimers and for obtaining DNA fragments of 200 bps or larger Otherwise Protocol II should be used to select desired DNA fragment size ex 200 800 bps Protocol I For samples containing lt 50 ng of DNA fragments or when large size DNA fragments are not a concern a Resuspend MQ Binding Beads by vortex b Add 1 2X 1 2 1 ratio resuspended beads to the DNA sample in a 0 2 ml PCR tube ex 60 ul of MQ beads to 50 ul of DNA solution Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times Cc 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 2 minutes if the magnetic stand is not suitable for the PCR tube transfer the beads solution to an appropriate tube or plate well that is compatible to the magnetic stand Carefully remove and discard the supernatant Caution Be careful not to disturb or discard the beads that contain DNA e Keep the 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 f Repeat Step e two times for a total of three washes Make sure that the ethanol is completely removed after the last wash O
7. nd Roche 454 platforms e Fast and straightforward size selection procedure which can be finished within 30 min No gels columns or centrifugation is needed e Efficient removal of primer dimers No need for further clean up steps e High recovery of targeted DNA fragments Higher than 85 recovery of input DNA fragments e Manual and automation friendly Scalable for use in single tube or 96 well plate formats PRINCIPLE amp PROCEDURE The EpiNext DNA Size Selection Kit contains all reagents required at each step of the workflow for DNA size selection The size of DNA fragments bound to MQ Binding Beads is based on the ratio of MQ beads to the DNA sample solution Optimization of MQ bead ratio to input DNA allows the removal of larger or smaller DNA fragments and recovery of desired target size DNA fragments 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 2014 02 27 Epigentek Group Inc All rights reserved Products are for research use only P 1059 j b co s g see WS DNA sample y Selected size of DNA binding to MQ beads Wash DNA Elution of DNA Fig 1 Workflow of the EpiNext DNA Size Selection Kit A B Fu Py Fu 505 y o amp 505 av a ee ae ay eae eel el Ie el 50 300 500 700 1000 3000 10380 bp 50 300 500 700 1000 3000 10380 bp Fig 2 Siz
8. nd round selection 0 8X 0 6 X 0 55X 0 5X 0 45X 0 4X 0 2X 0 2X 0 2X 0 2X 0 2X 0 15X The quality of the size selected DNA can be assessed by using an Agilent Bioanalyzer or comparable method with or without PCR amplification If fragments of lt 150 bp such as primer adaptor dimers are present in the sample or larger fragments than expected are present in the sample it is recommend to use 0 8X MQ Binding Beads ex add 16 ul of MQ Binding Beads to 20 ul of sample to remove them according to Protocol Il and Table 1 with optimization of the beads to DNA ratio The selected DNA fragments can be stored at 20 C until ready to use 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 2014 02 27 Epigentek Group Inc All rights reserved Products are for research use only P 1059 TROUBLESHOOTING Problem Possible Cause Suggestion Low yield of selected Insufficient amount of starting To obtain the best results the amount DNA 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 storage of the kit Ensure that the kit has not exceeded the expiration date The standard shelf life when stored properly is 6 months from date of receipt Unex
9. pected peak size Improper ratio of MQ beads to Check if the correct volume of MQ of Agilent Bioanalyzer DNA volume during size selection Beads is added to the DNA solution trace Presence of Proper ratios should remove the lt 150 bp adaptor fragments of unexpected peak size dimers or presence of _ i ae larger fragments than Over amplification of library PCR artifacts from over amplification of expected 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 Clean up 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 MeDIP Kit P 1038 EpiQuik Hydroxymethylated DNA Immunoprecipitation nMeDIP Kit P 1052 EpiQuik MeDIP Ultra Kit P 2002 EpiQuik Chromatin Immunoprecipitation ChIP Kit P 2003 EpiQuik Tissue Chromatin Immunoprecipitation ChIP Kit P 2014 EpiQuik Plant ChIP Kit P 2025 ChromaFlash One Step ChIP Kit P 2026 ChromaFlash One Step Magnetic ChIP kit P 202
10. pen the tube cap and air dry beads for 10 minutes while the tube is on the magnetic stand Resuspend the beads in 10 20 ul Elution Buffer and incubate at room temperature for 2 minutes to release the DNA from the beads i Capture the beads by placing the tube in the magnetic stand for 2 minutes or until the solution is completely clear ae 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 2014 02 27 Epigentek Group Inc All rights reserved Products are for research use only P 1059 j Transfer 10 20 ul of supernatant to a new 0 2 ml PCR tube for PCR amplification Protocol Il For samples containing gt 50 ng of DNA fragments or that require removal of large size DNA fragments a Resuspend MQ Binding Beads by vortex b Add resuspended MQ Binding Beads to the input DNA solution for the desired DNA size according to Table 1 Beads to DNA ratio in first round selection 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 2 minutes If the magnetic stand is not suitable for the PCR tube transfer the beads solution to an appropriate tube or plate well that is compatible to the magnetic stand Carefully transfer the supernatant containing DNA to a new tube Caution do not discard the
11. supernatant which contains the desired DNA fragment size Discard the beads that contain the unwanted large fragments e Add resuspended MQ Binding Beads to the supernatant for the desired DNA size according to Table 1 Beads to DNA ratio in second round size selection 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 Caution Be careful not to disturb or discard the beads that 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 g 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 10 20 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 10 20 ul of eluted DNA to a new 0 2 ml PCR tube for PCR amplification Table 1 Selection of desired DNA fragment size by optimized ratio of MQ beads to input DNA solution Desired DNA size bps 200 300 400 500 600 800 Beads to DNA ratio in first round selection Beads to DNA ratio in seco
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