EpiNext™ Bisulfite Sequencing Kit (Illumina)
Contents
1. Ligation a l LLA Amplification A ia Purification TT T T T roe TTT 50 300 s00 700 1500 10380 Ibp NGS Illumina Fig 2 Size distribution of library fragments Post bisulfite DNA library was prepared from 10 ng of input DNA using the EpiNext Bisulfite Sequencing Kit Fig 1 Workflow of the EpiNext Bisulfite Sequencing Kit ASSAY PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Input DNA Amount DNA amount can range from 500 pg to 1 ug per reaction An optimal amount is 100 ng to 200 ng per reaction Starting DNA may be in water or in a buffer such as TE DNA should be of high quality and relatively free of RNA RNAse can be used to remove RNA DNA Isolation You can use your method of choice for DNA isolation Epigentek offers a series of genomic DNA isolation kits for your convenience DNA Storage Isolated genomic DNA can be stored at 4 C or 20 C until use 1 Bisulfite DNA Modification 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 11 24 Epigentek Group Inc All rights reserved Products are for research use only P 1056 a Add 1 ml of Modification Buffer to 1 vial of Modification Powder to generate Modification Solution Mix by inverting and shaking the vial repeatedly for 3 4 min trace amount o2. 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 DNA Bisulfite Conversion P 1001 Methylamp DNA Modification KIt P 1026 BisulFlash DNA Modification 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 Prep Kit Illumina NGS Barcode P 1060 EpiNext NGS Barcode Index Set 12 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 13 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 11 24 Epigentek Group Inc All rights reserved Products are for research use only P 1056
3. EpiNext Bisulfite Sequencing Kit Illumina Base Catalog P 1056 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiNext Bisulfite Sequencing Kit Illumina is designed to carry out bisulfite conversion followed by a post bisulfite library preparation process for Illumina platform based bisulfite sequencing all in one kit Intended applications include whole genome bisulfite sequencing oxidative bisulfite sequencing reduced representative bisulfite sequencing and various other bisulfite based next generation sequencing techniques The optimized protocol and components of the kit allow the DNA to be bisulfite converted and fragmented simultaneously followed by quick non barcoded singleplexed and barcoded mutilplexed library construction using sub nanogram quantities of bisulfite converted DNA Starting Material and Input amount Starting materials can be genomic DNA isolated from various tissue cell samples such as fresh and frozen tissues cultured cells from a flask or microplate microdissection samples FFPE tissues plasma serum and body fluid samples etc DNA enriched from various enrichment reactions such as ChIP MeDIP hMeDIP or exon capture may also be used as starting materials DNA should be without any previous restriction digestion step Plasmid DNA can be used for bisulfite treatment with or without previous linearization as the kit allows for DNA denaturation status to remain during the entire DNA bisulfi
4. 72 C 20 sec Final Extension 72 C 2 min 1 PCR cycles may vary depending on the input DNA amount In general use 10 PCR cycles for 500 ng 14 cycles for 50 ng 18 cycles for 5 ng and 22 cycles for 1 ng DNA input Further optimization of PCR cycle number may be required 11 Clean Up of Amplified Library DNA a Resuspend MQ Binding Beads by vortex b 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 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 2 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 f Repeat Step 11e two times for total of three washes g Open the PCR tube cap and air dry beads for 10 minutes while the tube is on the magnetic stand h Resuspend the beads in 22 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 j Transfer 20 ul to a new 0 2 ml PCR tube Quality o
5. min at 75 C in a thermocycler without heated lid 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 11 24 Epigentek Group Inc All rights reserved Products are for research use only P 1056 8 Adaptor Ligation Prepare a reaction mix for adaptor ligation according to Table 4 Add the following reagents to a 0 2 ml PCR tube containing end repaired dA tailed DNA from Step 7 Table 4 Adaptor Ligation Component Volume End repaired dA tailed DNA from Step 7 15 ul 2X Ligation Buffer 17 ul T4 DNA Ligase 1 ul Adaptors 1 ul Total Volume 34 ul Mix and incubate for 10 min at 25 C in a thermocycler without heated lid Note 1 The pre annealed adaptors included in the kit are suitable for both non barcoded 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 9 Size Selection Clean up 9 1 Size Selection of Ligated DNA Optional Note If the starting DNA amount is less than 200 ng size selection is not recommended and alternatively clean up of li
6. P MeDIP hMeDIP or exon capture may also be used as starting materials PRINCIPLE amp PROCEDURE This kit includes all reagents required for a successful preparation of a DNA library by directly using bisulfite converted DNA generated from a small amount of input DNA 500 pg to 500 ng In this preparation DNA is bisulfite converted and fragmented to the appropriate length simultaneously during the bisulfite process The bisulfite treated DNA which is in single stranded form is then converted to dsDNA and directly used for ligation with BisDNA specific adaptors that are necessary for amplification and sequencing The fragments are size selected and purified using MQ binding beads which allows for quick and precise size selection of DNA Size selected DNA fragments are amplified using a high fidelity PCR Mix which ensures maximum yields from minimum amounts of starting material and provides highly accurate amplification of library DNA with low error rates and minimal bias 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 11 24 Epigentek Group Inc All rights reserved Products are for research use only P 1056 j bb co s g se K Input DNA Bisulfite Modification L LLLU peuuuuuur v dsDNA Conversion Fu s M E End Repair 405 ee a dA Tailing 304 204 Adaptor o OO
7. agnetic 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 Repeat Step 4e one time for a total of two washes Open the cap of the PCR tube 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 2 minutes or until the solution is completely clear Transfer clear solution to a new 0 2 ml PCR tube for end repair reaction 5 DNA End Repairing a Prepare end repair reaction in 0 2 ml PCR tube according to Table 2 110 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 11 24 Epigentek Group Inc All rights reserved Products are for research use only P 1056 Table 2 End Repair Reaction Component Volume Converted dsDNA from Step 4 11 12 ul 10X End Repair Buffer 2 ul End Repair Enzyme Mix 1 ul Distilled Water 5 6 ul Total Volume 20 ul b Mix and incubate for 30 min at 20 C in a thermocycler 6 Clean up of End Repaired DNA a Resuspend MQ Binding Beads by vortex b Add 36 ul of resuspended beads to the PCR tube of end repair reaction Mix thoroughly on a vortex mixer or by
8. and prevents handling errors as well as loss of valuable samples e Complete conversion The innovation reagent composition converts unmethylated cytosine into uracil at a level greater than 99 9 with negligible inappropriate or error conversions of methylcytosine to thymine lt 0 1 e High sensitivity efficiency and flexibility Adaptor ligation after bisulfite treatment eliminates loss of fragments and selection bias which enables input DNA to be as low as 0 5 ng The kit can be used for both non barcoded singleplexed and barcoded multiplexed DNA library preparation e Extremely convenient the kit contains all the required components for each step of the DNA library preparation process which are sufficient for bisulfite conversion ligation clean up size selection and library amplification thereby allowing the bisulfite DNA library preparation to be streamlined for the most reliable and consistent results e Minimal bias Ultra HiFi amplification enables achievement of reproducibly high yields of DNA libraries with minimal sequence bias and low error rates e Broad sample suitability Starting materials can be genomic DNA isolated from various tissue cell samples such as fresh and frozen tissues cultured cells from a flask or microplate microdissection samples paraffin embedded tissues biopsy samples embryonic cells plasma serum samples and body fluid samples etc DNA enriched from various enrichment reactions such as ChI
9. e kit Ensure that the kit has not exceeded the expiration Standard shelf life when stored properly is 6 months from date of receipt Unexpected peak size of Agilent Bioanalyzer trace Presence of lt 150 bp adaptor dimers or presence of larger fragments than expected Improper ratio MQ Binding Beads to DNA volume in size selection Check if the correct volume of MQ Binding Beads is added to DNA solution accordingly Proper ratios should remove the fragments with unexpected peak size Ex use 0 8X MQ Binding Beads to remove fragments below 150 bps or to remove fragments above 500 bps follow the protocol according to steps a l of 9 1 Size Selection Insufficient ligation Too much and too little input DNA may cause insufficient ligation which can shift 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 12 Printed 2014 11 24 P 1056 peak size of fragment population to be shorter or larger than expected Make sure that ligation reaction 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
10. ed by running a gel Ensure that RNA is removed by Rnase treatment Too little DNA or too much DNA i e lt 500 pg or gt 1 yg Increase or decrease input DNA to within the correct range or to the optimal amount of 100 200 ng Temperature or thermocycling condition is incorrect Check for appropriate temperature or thermocycling conditions Insufficient DNA clean up Ensure that 30 ul of Desulphonation Solution is added into every 1 ml of 90 ethanol in Step 2c Elute contains little or no DNA Poor input DNA quality degraded Check if DNA is degraded by running a gel DNA Binding Solution is not added into the sample Ensure that DNA Binding Solution is added in Step 2a Concentration of ethanol solution used for DNA clean up is not correct Use 90 ethanol for DNA clean up Sample is not completely passed through the filter membrane of column Centrifuge for 1 min at 12 000 rpm or until the entire sample has passed through the filter membrane Low yield of library Insufficient amount of bisulfite DNA To obtain the best results the optimized amount of input DNA for bisulfite treatment should be 100 200 ng Improper reaction conditions at each reaction step Check if the reagents are properly added and incubation temperature and time are correct at each reaction step including Adaptor Ligation Size Selection and Amplification Improper storage of th
11. es Methylamp MS qPCR Fast kit Cat P 1028 for real time MS PCR Both positive primers b actin component of kit Cat No P 1028 and negative primers GAPDH component of kit Cat No P 1029 are also separately available for checking conversion efficiency 3 dsDNA Conversion Reaction a Prepare dsDNA Conversion reaction in a 0 2 ml PCR tube according to Table 1 Table 1 dsDNA Conversion Component Volume Bisulfite DNA 10 ul 100 200 ng input DNA 5X Conversion Buffer 4ul Conversion Primer 2 ul Distilled water 3 ul Total Volume 19 pl Mix and incubate for 5 min at 95 C in a thermocycler followed by 5 min at 4 C or on ice Add 1 ul of Conversion Enzyme Mix to the reaction tube Mix and incubate for 60 min at 37 C ina thermocycler Note The optimal amount of input DNA should be 100 ng to 200 ng and eluted volume after bisulfite treatment should be lt 20 ul 4 Clean Up of Converted dsDNA j Resuspend MQ Binding Beads by vortex Add 36 ul of resuspended beads to the PCR tube of dsDNA conversion reaction Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times Incubate for 10 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 2 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 m
12. f the prepared library can be assessed using an Agilent Bioanalyzer or other comparable methods 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 e g dilute amplified library DNA to 20 ul with TE and then add 20 ul of MQ Binding Beads according to sub steps a through j of Step 11 Clean up of Amplified Library DNA To remove fragments above 500 bps follow sub steps a through of Step 9 1 Size Selection of Ligated DNA The prepared DNA library can be quantified using various DNA library quantification methods The prepared library DNA can be stored at 20 C until ready to use for sequencing TROUBLESHOOTING 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 11 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 11 24 Epigentek Group Inc All rights reserved Products are for research use only P 1056 Problem Possible Cause Suggestion DNA is poorly modified Poor DNA quality DNA is severely degraded Check if the sample DNA 260 280 ratio is between 1 8 1 9 and if DNA is degrad
13. f undissolved Modification Powder may remain which is normal as Modification Powder is saturated in solution For each 0 2 ml PCR tube add 150 ul of the mixed Modification Solution followed by adding 1 5 pl of sample DNA Note Check if the sample DNA volume is large and the concentration is lower than 5 ng ul If so it is recommended to concentrate DNA using Epigentek s DNA Concentrator Kit Cat No P 1006 prior to bisulfite treatment Prepared Modification Solution can be stored at 20 C for up to 2 weeks without significant loss of efficiency For the best results the mixed solution should be used immediately Tightly close the PCR tubes and place them in a thermocycler with heated lid Program and run the thermocycler according to the following 95 CT 5 min 65 CT 30 min 95 CT 5 min 65 CT 30 min 95 CT 5 min 65 60 min Hold 18 20 CT up to 6h Meanwhile insert the number of F Spin Columns into F Collection Tubes as needed by your experiment 2 Modified DNA Clean Up a Add 250 ul of DNA Binding Solution to each column Then transfer the samples from each PCR tube from Step 1 to each column containing the DNA Binding Solution Centrifuge at 12 000 rpm for 45 sec Remove columns from collection tubes and discard the flowthrough Place columns back into collection tubes Add 250 ul of 90 ethanol to each column Centrifuge at 12 000 rpm for 45 sec Prepare final Desulphonation Buffer by adding 30 ul of Desulph
14. gated DNA can be performed prior to PCR amplification according to the Step 9 2 protocol Clean Up of Ligated DNA 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 2 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 of 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 2 minutes Carefully remove and discard the supernatant 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 9 1g one time for a 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 s
15. ips PCR tubes or plates 1 5 ml microcentrifuge tubes 100 Ethanol Distilled water oO o O o o o O Oo GENERAL PRODUCT INFORMATION DNA sample Quality Control Each lot of EpiNext Bisulfite Sequencing 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 Bisulfite Sequencing Kit Illumina is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The EpiNext Bisulfite Sequencing Kit Illumina and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW DNA methylation occurs by the covalent addition of a methy
16. l group CH3 at the 5 carbon of the cytosine ring resulting in 5 methylcytosine 5 mC DNA methylation is essential in regulating gene expression in nearly all biological processes including development growth and differentiation Alterations in DNA methylation have been demonstrated to cause a change in gene expression For example hypermethylation leads to gene silencing or decreased gene expression while hypomethylation activates genes or increases gene expression Aberrant DNA methylation is also associated with pathogenesis of diseases such as cancer autoimmune disorders and schizophrenia Thus genome wide analysis of DNA methylation could provide valuable information for discovering epigenetic markers used for disease diagnosis and potential targets used for therapeutics 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 11 24 Epigentek Group Inc All rights reserved Products are for research use only P 1056 Several methods such as whole genome bisulfite sequencing WGBS or reduced representation bisulfite sequencing RRBS are currently used for genome wide DNA methylation analysis These methods convert unmethylated cytosine to uracil while 5 mC remains unmodified by the bisulfite treatment This allows epigenetic differences to be interpreted as genetic differences which can then be detected by sequencing at sing
17. le base resolution and on a genome wide scale However traditional methods to achieve this still do not have practical use because 1 such methods require large amounts of DNA gt 1 ug as input material which is difficult to prepare from limited biological samples such as tumor biopsy samples early embryos embryonic tissues and circulating DNA 2 such methods require that DNA is first sheared and then ligated to adaptors followed by bisulfite conversion post ligation bisulfite conversion This procedure causes most of the DNA fragments contained in the adaptor DNA fragment constructs to be broken and thereby form mono tagged templates that will be removed during library enrichment Thus incomplete coverage and bias occur when performing whole genome bisulfite sequencing and 3 such methods are time consuming 2 days To overcome the weaknesses of these methods Epigentek offers the EpiNext Bisulfite Sequencing Kit Illumina The kit has the following features e Innovative method Allows for simultaneous bisulfite conversion and size appropriate DNA fragmentation The bisulfite DNA can be directly ligated to adaptors thereby eliminating the possibility of breaking adaptor ligated fragments which often occurs with traditional WGBS and RRBS methods e Fast and streamlined procedure the procedure from DNA bisulfte treatment to PCR amplification can be finished within the same day lt 8 h Gel free size selection purification saves time
18. onation Solution to every 1 ml of 90 ethanol and mix Add 100 ul of the final Desulphonation Buffer Desulphonation Solution and 90 ethanol mixture to each column Allow columns to sit for 15 min at room temperature then centrifuge at 12 000 rpm for 45 sec Remove columns from collection tubes and discard the flowthrough Place columns back into collection tubes Add 250 ul of 90 ethanol to each column Centrifuge at 12 000 rpm for 45 sec Remove columns from collection tubes and discard the flowthrough Place columns back into collection tubes Add 250 ul of 90 ethanol to each column again and centrifuge at 12 000 rpm for 45 sec Insert each column into a new 1 5 ml tube Add 10 ul of Elution Solution directly to each column s filter membrane Centrifuge at 12 000 rom for 60 sec to elute converted DNA Modified DNA is now ready to use for post bisulfite DNA library preparation or storage at or below 20 C for up to 3 months The peak size of converted DNA is 250 300 bps 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 Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 11 24 P 1056 Note To ensure the DNA is properly modified we recommend checking the bisulfite treated DNA by real time methylation specific PCR MS PCR For your convenience and the best results Epigentek provid
19. pipetting up and down at least 10 times C Incubate for 10 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 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 90 ethanol to the tube Incubate at room temperature for 1 min and then carefully remove and discard the ethanol f Repeat Step 6e one time for a total of two washes g Open the cap of the PCR tube and air dry beads for 10 minutes while the tube is on the magnetic stand h Resuspend the beads in 12 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 j Transfer clear solution to a new 0 2 ml PCR tube for dA tailing reaction 7 DNA dA Tailing a Prepare the reaction mix for dA tailing according to Table 3 Add the following reagents to a 0 2 ml PCR tube containing end repaired DNA from step 6 Table 3 dA Tailing Reaction Component Volume End repaired DNA from Step 6 12 ul 10X dA tailing Buffer 1 5 ul Klenow Fragment 3 5 exo 1 ul Distilled Water 0 5 ul Total Volume 15 pl b Mix and incubate for 30 min at 37 C followed by 10
20. solution is completely clear Transfer 11 ul clear solution to a new 0 2 ml PCR tube for PCR amplification 10 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 pl HiFi Master Mix 2X 12 5 ul Primer U 1 ul Primer 1 ul Adaptor Ligated DNA 10 5 ul Total Volume 25 pl 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 of Primer I Program the PCR Reactions Place the reaction plate in the PCR instrument and set the PCR conditions as follows 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 10 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 11 24 Epigentek Group Inc All rights reserved Products are for research use only P 1056 Cycle Step Temp Cycle Activation 98 C 30 sec 1 98 C 20 sec Cycling 55 C 20 sec Variable
21. tand for 4 minutes or until the solution is completely clear 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 Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 11 24 P 1056 Transfer clear solution to a new 0 2 ml PCR tube for PCR amplification 9 2 Clean Up of Ligated DNA j Resuspend MQ Binding Beads by vortex 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 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 2 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 the ethanol Repeat Step 9 2e 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 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 2 minutes or until the
22. te conversion process and direct ligation of adaptors to bisulfite DNA Input amount of DNA can be from 0 5 ng to 1 ug For optimal preparation the input amount should be 100 ng to 200 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 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 11 24 Epigentek Group Inc All rights reserved Products are for research use only P 1056 KIT CONTENTS Component 12 reactions 24 reactions Storage Cat P 1056 12 Cat P 1056 24 Upon Receipt Modification Buffer 3 ml 6 ml RT Modification Powder 2 vials 4 vials RT DNA Binding Solution 6 ml 12 ml RT Desulphonation Solution 70 ul 140 ul RT Elution Solution 0 5 ml 1ml RT F Spin Column 15 30 RT F Collection Tube 15 30 RT 5X Conversion Buffer 50 ul 100 ul 20 C Conversion Enzyme Mix 15 ul 30 ul 20 C Conversion Primer 26 ul 52 ul 20 C 10X End Repair Buffer 40 ul 80 ul 20 C End Repair Enzyme Mix 25 ul 50 ul 20 C 10X dA Tailing Buffer 40 ul 80 ul 20 C Klenow Fragment 3 5 exo 15 ul 30
23. ul 20 C 2X Ligation Buffer 250 ul 500 pl 20 C T4 DNA Ligase 15 ul 30 ul 20 C Adaptors 50 uM 15 ul 30 ul 20 C MQ Binding Beads 1 8 ml 3 6 ml 4 C 2X HiFi PCR Master Mix 160 ul 320 ul 20 C Primer U 10 uM 15 ul 30 ul 20 C Primer 10 uM 15 ul 30 ul 20 C Elution Buffer 1000 ul 2000 ul 20 C 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 20 C immediately 5X Conversion Buffer Conversion Enzyme Mix Conversion Primer 10X End Repair Buffer End Repair Enzyme Mix 10X dA Tailing Buffer Klenow Fragment 3 5 exo 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 O Agilent Bioanalyzer or comparable method to assess the quality of DNA library O Thermocycler 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 2 Printed 2014 11 24 P 1056 Centrifuge including desktop centrifuge up to 14 000 rpm Magnetic stand 96 well format Pipettes and pipette t
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