EpiNext™ DNA Purification HT System
Contents
1. EpiNext DNA Purification HT System Base Catalog P 1063 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiNext DNA Purification HT System utilizes magnetic bead technology for high throughput DNA or PCR amplicon purification and DNA size selection The system can also be used for concentrating DNA and is suitable for selectively capturing DNA fragments or PCR amplicons that are 100 bps or larger in size A total of 96 P 1063 04 or 192 P 1063 08 standard purifications use 0 5 ug of DNA in 20 ul of solution or 48 P 1063 04 or 96 P 1063 08 standard DNA size selections use 50 ul of input DNA solution can be performed with the bead volume provided in the system 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 2015 10 02 Epigentek Group Inc All rights reserved Products are for research use only P 1063 PRODUCT CONTENTS Component Cat P 1063 04 Cat P 1063 08 Cat P 1062. 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 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 7 Printed 2015 10 02 P 1063 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 2027 ChromaFlash High Sensitivity ChIP Kit 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 P 1059 EpiNext DNA Size Selection Kit 110 Bi County Blvd Ste 122 Farmingd
3. or for storage at 20 C after capping the tube Note For size selection of DNA fragments we recommend using the protocol from the EpiNext DNA Size Selection Kit Cat No P 1059 which includes the MQ Binding Beads TROUBLESHOOTING Problem Possible Cause Suggestion To obtain the best results the amount of input DNA should be gt 10 ng Low yield of purified Insufficient amount of starting DNA DNA Insufficient purity of starting DNA Ensure that RNA is removed by RNAse treatment before starting purification protocol Ensure that the kit has not exceeded the expiration date The standard shelf life when stored properly is 6 months from date of receipt Improper storage of the kit Check if the correct volume of MQ Binding Beads is added to the DNA Unexpected peak size of Agilent Bioanalyzer Improper ratio of MQ beads to DNA volume during size selection trace Presence of lt 150 bp adaptor dimers or presence of larger fragments than expected during the DNA library preparation solution Proper ratios should remove the fragments of unexpected peak size 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 Clean up P 1003 FitAmp General Tissue Section DNA Isolation Kit
4. 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 g Air dry beads for 5 minutes at room temperature while the plate is on the magnetic stand to ensure all traces of ethanol are removed 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 2015 10 02 Epigentek Group Inc All rights reserved Products are for research use only P 1063 Note Take care not to over dry the bead spot an over dried bead spot appears cracked as this will significantly decrease elution efficiency h Resuspend the beads in 10 20 ul DNA 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 j Transfer 10 20 ul of supernatant to a new 0 2 ml PCR tube for downstream use
5. 3 64 Cat P 1063 X4 Storage Upon Receipt MQ Binding 4 ml 8 ml 64 ml 480 ml 4 C Beads User Guide 1 1 1 1 RT SHIPPING amp STORAGE The product is shipped at room temperature Upon receipt Store the following components at 4 C MQ Binding Beads Store all other components at room temperature The beads are stable for at least 6 months from the shipment date when stored properly MATERIALS REQUIRED BUT NOT SUPPLIED O Vortex mixer Magnetic stand 96 well format Pipettes and pipette tips 0 2 ml PCR tubes for single tube format 96 well round bottom plate or 96 well cycling plate 80 ethanol DNA sample 0 OF O 0 0 0 O DNA elution buffer DNase RNase free water or TE buffer GENERAL PRODUCT INFORMATION Quality Control Each lot of EpiNext DNA Purification HT System 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 o
6. DNA marker Hyperladder 50 bp ladder Bioline using the EpiNext DNA Purification HT System with varying ratios of beads from 0 9X to 2 1X O9X 1 2X 1 8X 21X O9X 1 2X 1 8X 21X AMPure XP beads were used as the control M Marker size of Agilent DNA ChIP kit 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 Printed 2015 10 02 100 ZS 90 A 3 80 5 70 lt gt 2 60 g se 50 AMPure XP EpiNext Beads Fig 3 Percentage of recovery of input DNA fragments by AMPure XP beads vs EpiNext beads 780 ng of DNA marker Hyperladder 50 bp ladder Bioline were purified using the EpiNext DNA Purification HT System at 1 8X Agencourt AMPure XP beads 1 8X were used as the control Recovery yield of 100 bp or higher DNA fragments was quantified with an Agilent Bioanalyzer 2100 ASSAY PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials e DNA isolated from various tissues or cell samples 0 2 ng 1000 ng optimized 20 500 ng per preparation e DNA fragments enriched from a ChIP reaction MeDIP hMeDIP reaction or exon capture 0 2 ng 100 ng e cDNA or dsDNA converted from reverse transcription of RNA or bisulfite treatment of DNA e PCR amplicons R
7. NAse can be used to remove RNA and DNA should be eluted in DNase RNase free water For the magnetic stand used for capturing 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 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 A h Printed 2015 10 02 Epigentek Group Inc All rights reserved Products are for research use only Ke P 1063 DNA Purification 96 Well Format a b 9 Resuspend MQ Binding Beads by vortex Add 2X 2 1 ratio resuspended beads to the DNA sample in 96 well plate ex 40 ul of MQ beads to 20 ul of DNA solution 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 the beads Put the plate on an EpiMag HT Magnetic Separator or an appropriate magnetic stand until the solution is clear about 2 minutes if the magnetic stand is not suitable for the plate transfer the beads solution to an appropriate 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 Keep the plate in the magnetic stan
8. ale 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 2015 10 02 Epigentek Group Inc All rights reserved Products are for research use only P 1063
9. d and add 200 ul of freshly prepared 80 ethanol to each well of the plate Incubate at room temperature for 1 min and then carefully remove and discard the ethanol Repeat Step e two times for a total of three washes Make sure that the ethanol is completely removed after the last wash Air dry beads for 5 minutes at room temperature while the plate is on the magnetic stand to ensure all traces of ethanol are removed Note Take care not to over dry the bead spot an over dried bead spot appears cracked as this will significantly decrease elution efficiency Resuspend the beads in 10 20 ul DNA elution buffer and incubate at room temperature for 2 minutes to release the DNA from the beads Capture the beads by placing the plate in the magnetic stand for 2 minutes or until the solution is completely clear Transfer 10 20 ul of supernatant to a new 0 2 ml PCR plate for immediate use or for storage at 20 C after tightly capping the PCR plate Single PCR Tube Format a Resuspend MQ Binding Beads by vortex b Add 2X 2 1 ratio resuspended beads to the DNA sample in a 0 2 ml PCR tube ex 40 ul of MQ beads to 20 ul of DNA solution 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 the beads d Put the PCR tube on an EpiMag HT Magnetic Separator or an appropriate magnetic stand until the solution is clear about 2 minutes If the magnetic
10. min for 96 samples and is highly amenable to a variety of automation platforms No gels filtration centrifugation or columns are needed e Efficient clean up Removal of excess primers adaptors nucleotides salts enzymes and PCR inhibiting substances such as polysaccharides polyphenols lipids and dyes e High recovery of DNA Higher than 98 recovery of input DNA e Manual and automation friendly Scalable for use in single tube or 96 well plate formats PRINCIPLE amp PROCEDURE The EpiNext DNA Purification HT System contains an optimized MQ binding bead solution which allows DNA or PCR amplicons to bind tightly to the beads Excess primers adaptors nucleotides salts enzymes and PCR inhibiting substances can be removed by simply washing the beads Optimization of MQ bead ratio to input DNA allows DNA size selection by 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 2015 10 02 Epigentek Group Inc All rights reserved Products are for research use only P 1063 Binding of DNA to MQ beads Wash DNA Elution of DNA Fig 1 Workflow of the EpiNext DNA Purification HT System M AMPure XP Beads EpiNext Beads 100 Fig 2 Gel picture after purification of 800 ng of
11. r 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 product when using that product 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 2 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2015 10 02 Epigentek Group Inc All rights reserved Products are for research use only P 1063 Usage Limitation The EpiNext DNA Purification HT System is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The EpiNext DNA Purification HT System and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW Obtaining high recovery of purified DNA or selected DNA fragments is critical for downstream applications that use DNA samples including PCR sequencing cloning microarray and DNA fragment analysis regardless of the platform used The EpiNext DNA Purification HT System utilizes magnetic bead technology for high throughput DNA or PCR amplicon purification and DNA size selection The System has the following features e Optimized buffer chemistries Complete separation of DNA or PCR amplicons It can also be used for DNA size selection based on the ratio of beads to DNA sample volume e Fast and straightforward The entire procedure can be finished in just 30
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