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EpiNext™ High-Sensitivity Bisulfite-Seq Kit

1. 22 cycles is for 10 ng of input DNA PCR cycles may vary depending on the input DNA amount In general use 19 cycles for 50 ng 24 cycles for 1 ng and 26 cycles for 0 2 ng of input DNA Further optimization of PCR cycle number may be required 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 Web www epigentek com Printed 2014 05 30 Epigentek Group Inc All rights reserved Products are for research use only P 1056A 8 Clean Up of Amplified Library a Resuspend MQ Binding Beads by vortex b Add exactly 20 ul 0 8X of resuspended beads to the amplified library 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 3 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 150 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 Remove tube from magnet Add 150 ul of freshly prepared 90 ethanol to the tube to resuspend the beads Put the PCR tube back in the magnetic stand Incubate at room temperature for 1 min and then carefully remove and discard
2. 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 0 2 ng to 200 ng per reaction An optimal amount is 10 ng 50 ng per reaction Starting DNA may be in water or in a buffer such as TE DNA should be 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 DNA Fragment Purification We recommend using Epigentek s EpiMag HT 96 Well Magnetic Separator Cat No Q10002 for DNA fragment purification with MQ Binding Beads 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 1 Bisulfite DNA Modification 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 a trace amount of 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 10 ul of sample DNA 20 100 ng Note Check if the sample DNA volu
3. EpiNext High Sensitivity Bisulfite Seq Kit Illumina Base Catalog P 1056A PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiNext High Sensitivity Bisulfite Seq Kit is designed to prepare bisulfite converted DNA libraries for Illumina platform based bisulfite sequencing including whole genome bisulfite sequencing oxidative bisulfite sequencing reduced representation bisulfite sequencing and other bisulfite next generation sequencing The optimized protocol and components of the kit allow subnanogram DNA to be used for simultaneous bisulfite conversion and fragmentation followed by non barcoded singleplexed and barcoded multiplexed library construction in less than 7 hours Starting Material and Input amount Starting materials can be genomic DNA isolated from various tissue cell samples such as fresh and frozen tissue cultured cells from a flask or microplate microdissection samples FFPE tissue 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 material 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 bisulfite conversion process and direct ligation of adaptors to bisulfite DNA Input amount of DNA can be from 0 2 ng to 200 ng For op
4. Prepare dsDNA Conversion reaction in 0 2 ml PCR tube according to Table 1 Table 1 dsDNA Conversion Converted DNA from Step 2 10 ul 10 50 ng input DNA 5X Reaction Buffer 4ul Adaptor F 10 uM 2 ul Reaction Enzyme Mix 4 ul Total volume 20 ul If converted DNA volume is less than 10 ul add distilled water to make the total volume 20 ul Mix and incubate for 55 min at 37 C in a thermocycler without heated lid make sure to set lid temperature to 25 C 4 Clean Up of dsDNA Note To ensure the correct ratio of MQ Binding Beads to sample solution during DNA clean up make sure that any bead solution stuck on the outside of the pipette tip is removed before adding beads into the sample vial Resuspend MQ Binding Beads by vortex Add exactly 20 ul of resuspended beads to the PCR tube of dsDNA synthesis reaction Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times 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 Web www epigentek com Printed 2014 05 30 Epigentek Group Inc All rights reserved Products are for research use only P 1056A j 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 di
5. NA sample 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 Sensitivity Bisulfite Seq Kit is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The EpiNext High Sensitivity Bisulfite Seq Kit and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW DNA methylation occurs by the covalent addition of a methyl 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 hypome
6. are size selected and purified using MQ Binding Beads followed by amplification with 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 minimum 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 05 30 Epigentek Group Inc All rights reserved Products are for research use only P 1056A Ful 504 40 305 204 104 lt Input DNA Bisulfite Conversion dsDNA Synthesis Adaptor Ligation Amplification Index Addition NGS Illumina 3000 10380 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 Printed 2014 05 30 Epigentek Group Inc All rights reserved Products are for research use only ACATGGTGAAACCCCAT ACATGGTGAAACCCCATI FU 604 504 404 304 204 TGGTGAAACCCCATI TGGTGAAACCCCAT Fig 1 Workflow of the EpiNext High Sensitivity Bisulfite Seq Kit B 50 300 500 700 1000 3000 10380 bp Fig 2 Size distribution of library fragments Post bisulfite DNA libraries were prepared from human placenta DNA using the EpiNext High Sensitivity Bisulfite Seq Kit A 10 ng B 50 ng Page 5 P 1056A
7. d 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 High Sensitivity Bisulfite Seq Kit Illumina This 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 currently used WGBS and RRBS methods e Fast and streamlined procedure The procedure from DNA bisulfite treatment to ready to use library DNA can be completed within 6 hours and 30 minutes e Complete conversion Completely converts unmethylated cytosine into uracil gt 99 with negligible inappropriate or error conversions of methylcytosine to thymine e High sensitivity and efficiency Innovative adaptor ligation of bisulfite DNA eliminates loss of fragments and selection bias which enables input DNA to be as low as 0 2 ng making it ideal for methylation profiling of precious limited samples The kit can be used f
8. eaction conditions at each reaction step Check if the reagents are properly added and incubation temperature and time are correct at each reaction step including dsDNA Conversion Library Synthesis and Library Amplification and Indexing 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 Presence of lt 150 bp adaptor dimers Improper ratio of MQ Binding Beads to DNA volume in size selection Check if the correct volume of MQ Binding Beads is added to DNA solution accordingly Use 0 8X MQ Binding Beads to remove fragments below 150 bps Insufficient ligation Too little input DNA and too much adaptors may cause insufficient ligation and adaptor dimers Make sure that ligation reaction is properly processed with the proper amount of input DNA and adaptors Over amplification of library PCR artifacts from over amplification of library may cause increased adaptor dimers Make sure to use proper PCR cycles to avoid this problem RELATED PRODUCTS DNA Isolation and Cleanup P 1003 P 1004 P 1006 P 1007 P 1009 P 1017 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 FitAmp General Tissue Sect
9. final desulphonation buffer Desulphonation Solution and 90 ethanol mixture to each column Allow columns to sit for 10 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 5 ul of Elution Solution directly to each column s filter membrane Centrifuge at 12 000 rpm for 60 sec to elute converted DNA Converted 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 150 300 bps Note If the amount of input DNA was gt 10 ng you may ensure the DNA is properly converted We recommend checking the bisulfite treated DNA by real time methylation specific PCR MS PCR For your convenience and the best results Epigentek provides Methylamp MS qPCR Fast Kit Cat No 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
10. ion DNA Isolation Kit FitAmp Plasma Serum DNA Isolation Kit DNA Concentrator Kit FitAmp Gel DNA Isolation Kit FitAmp Paraffin Tissue Section DNA Isolation Kit FitAmp Urine DNA Isolation Kit Page 11 Printed 2014 05 30 P 1056A Q10002 EpiMag HT 96 Well Magnetic Separator 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 PCR Analysis P 1028 Methylamp MS qPCR Fast Kit DNA Library Preparation P 1051 EpiNext DNA Library Preparation Kit Illumina P 1053 EpiNext High Sensitivity DNA Library Preparation Kit Illumina P 1059 EpiNext DNA Size Selection Kit P 1063 EpiNext DNA Purification HT System NGS Barcode P 1060 EpiNext NGS Barcode Index Set 12 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 12 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 05 30 Epigentek Group Inc All rights reserved Products are for research use only P 1056A
11. me is large and if the concentration is less 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 C5 min 65 TC 90 min Meanwhile insert the number of F Spin Columns into F Collection Tubes as needed by your experiment 2 Converted DNA Clean Up a Add 300 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 b Add 250 ul of 90 ethanol to each column Centrifuge at 12 000 rpm for 45 sec 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 05 30 P 1056A Prepare final desulphonation buffer by adding 30 ul of Desulphonation Solution to every 1 ml of 90 ethanol and mix Add 100 ul of the
12. or both non barcoded singleplexed and barcoded multiplexed DNA library preparation e Extremely convenient The kit contains all required components for each step of DNA library preparation 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 Board sample suitability Starting materials can be genomic DNA isolated from various tissue cell samples such as fresh and frozen tissue cultured cells from a flask or microplate microdissection samples paraffin embedded tissue biopsy embryonic cells plasma serum samples and body fluid samples etc DNA enriched from various enrichment reactions such as ChIP MeDIP hMeDIP or exon capture may be also used as starting material PRINCIPLE amp PROCEDURE This kit includes all reagents required for successfully preparing a library directly using bisulfite DNA generated from a tiny amount of input DNA In this preparation DNA is simultaneously bisulfite converted and fragmented to the appropriate length during the bisulfite process The bisulfite treated DNA which is in single stranded form is then simultaneously converted to dsDNA and adaptor ligated The ligated fragments
13. prior to use SHIPPING amp STORAGE The kit is shipped on frozen ice packs at 4 C Upon receipt 1 Store the following components at 20 C immediately 5X Reaction Buffer Reaction Enzyme Mix Adaptor F Adaptor S 2X HiFi PCR Master Mix Primer U Primer I and Elution Buffer 2 Store the following components at 4 C MQ Binding Beads 3 Store all other components at room temperature away from light MATERIALS REQUIRED BUT NOT SUPPLIED Vortex mixer Agilent Bioanalyzer or comparable method to assess the quality of the DNA library Thermocycler Centrifuge including desktop centrifuge up to 14 000 rpm Oo OF 0 0 OQ 96 well format magnetic stand e g EpiMag HT 96 Well Magnetic Separator Epigentek s Cat No Q10002 O Pipettes and pipette tips 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 05 30 P 1056A PCR tubes or plates 1 5 ml microcentrifuge tubes 100 Ethanol Distilled or Deionized water oO o o O o GENERAL PRODUCT INFORMATION Quality Control Each lot of EpiNext High Sensitivity Bisulfite Seq 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 D
14. s degraded by running gel Ensure that RNA is removed by RNase treatment Too little DNA or too much DNA Increase or decrease input DNA to within i e lt 5 pg or gt 200 ng the correct range or to the optimal amount of 10 50 ng Temperature or thermal cycling Check for appropriate temperature or condition is incorrect thermal cycling conditions Insufficient DNA clean up Ensure that 30 ul of Desulphonation Solution is added into every 1 ml of 90 ethanol in Step 2c 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 Web www epigentek com Printed 2014 05 30 Epigentek Group Inc All rights reserved Products are for research use only P 1056A 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 10 50 ng Improper r
15. scard the beads that contain DNA Keep the PCR tube in the magnetic stand and add 150 ul of freshly prepared 90 ethanol to the tube Incubate at room temperature for 1 min and then carefully remove and discard the ethanol Repeat Step 4e two times for a total of three washes Open the cap of the PCR tube and air dry beads for 2 3 minutes while the tube is on the magnetic stand Resuspend the beads in 10 5 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 10 ul of clear solution to a new 0 2 ml PCR tube for library synthesis 5 Library Synthesis a b Prepare library synthesis reaction in a 0 2 ml PCR tube according to Table 2 Table 2 Library Synthesis dsDNA from Step 4 10 ul 5X Reaction Buffer 4ul Adaptor S 10 uM 2 ul Total volume 16 ul Mix and incubate for 2 min at 98 C in a thermocycler without heated lid make sure to set lid temperature to 25 C followed by incubation on ice for 2 min Add 4 ul of Reaction Enzyme Mix and then incubate at 37 C for 60 min in a thermocycler without heated lid 6 Clean Up of Synthesized Library a Resuspend MQ Binding Beads by vortex b Add exactly 20 ul of resuspended beads to the tube of library synthesis reaction Mix thoroughly on a vortex mixer or by pipetting up and down at lea
16. st 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 Remove tube from magnet Add 150 ul of freshly prepared 90 ethanol to the tube to resuspend the beads Put the PCR tube back in the magnetic stand Incubate at room temperature for 1 min and then carefully remove and discard the ethanol f Keep the PCR tube in the magnetic stand and add 150 ul of freshly prepared 90 ethanol to the tube Incubate at room temperature for 1 min and then carefully remove and discard the ethanol g Repeat Step 6f one more time for a total of three washes h Open the cap of the PCR tube and air dry beads for 2 3 minutes while the tube is on the magnetic stand 110 Bi County Blvd Ste 122 Farmingdale NY 1 1735 i i 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 05 30 Epigentek Group Inc All rights reserved Products are for research use only P 1056A i Resuspend the beads in 11 ul Elution Buffer and incubate at room temperature for 2 minutes to release the DNA from the beads j Capture the beads by placing the tube in the magnetic stand for 2 minutes or until the solution is completely clear k Transfer 10 5 ul of clear solution
17. the ethanol g Repeat Step 8f one more time for a total of three washes h Open the PCR tube cap and air dry beads for 2 3 minutes while the tube is on the magnetic stand i Resuspend the beads in 10 ul Elution Buffer and incubate at room temperature for 2 minutes to release the DNA from the beads j Capture the beads by placing the tube in the magnetic stand for 2 3 minutes or until the solution is completely clear k Transfer 10 ul to a new 0 2 mI PCR tube for immediate use or store at 20 C until ready to use for sequencing Note 1 Quality of the prepared library can be assessed using an Agilent Bioanalyzer or comparable method Library fragments should have the correct size distribution ex 250 bps at peak size without adaptors or adaptor dimers about 127 bps 2 To check the size distribution dilute library with water if necessary and apply it to an Agilent high sensitivity chip If there is the presence of lt 150 bp adaptor dimers it is recommend to use 0 8X MQ Binding Beads to remove fragments below 150 bps 3 The amount of indexed library can be quantified using qPCR Qubit or Picogreen assays 4 Each indexed library can be combined in equal amounts to form multiplexed libraries for sequencing TROUBLESHOOTING Problem Possible Cause Suggestion DNA is poorly Poor DNA quality DNA is severely Check if the sample DNA 260 280 ratio is converted degraded between 1 8 1 9 and if DNA i
18. thylation 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 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 single base resolution and on a genome wide scale However traditional methods to achieve this still do not 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 Web www epigentek com Printed 2014 05 30 Epigentek Group Inc All rights reserved Products are for research use only P 1056A 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 ligate
19. timal preparation the input amount should be 10 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 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 Web www epigentek com Printed 2014 05 30 Epigentek Group Inc All rights reserved Products are for research use only P 1056A KIT CONTENTS Component 12 reactions 24 reactions Storage Cat P 1056A 12 Cat P 1056A 24 Upon Receipt Modification Buffer 3 ml 6 ml RT Modification Powder 2 vials 4 vials RT DNA Binding Solution 5 ml 10 ml RT Desulphonation Solution 50 ul 100 ul RT Elution Solution 0 5 ml 1 ml RT F Spin Column 15 30 RT F Collection Tube 15 30 RT 5X Reaction Buffer 100 ul 200 ul 20 C Reaction Enzyme Mix 100 ul 200 ul 20 C Adaptor F 10 uM 28 ul 56 ul 20 C Adaptor S 10 uM 28 ul 56 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 I 10 pM 15 ul 30 ul 20 C Elution Buffer 500 ul 1000 ul 20 C User Guide 1 1 RT Spin the solution down to the bottom
20. to a new 0 2 ml PCR tube for library amplification and indexing 7 Library Amplification and Indexing a Prepare the PCR Reactions Thaw all reaction components including master mix and primer solution 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 Table 3 Table 3 Library Amplification and Indexing Component Size pl HiFi Master Mix 2X 12 5 ul Primer U 1 ul Primer I or barcode index 1 ul Synthesized library DNA from 10 5 ul Step 6 Total Volume 25 ul Important Note 1 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 to generate each indexed library You can also add user defined barcodes Illumina compatible instead of Primer I 2 Each indexed library can be combined in equal amounts to form multiplexed libraries for sequencing 3 The amount of indexed library can be quantified using qPCR Qubit or Picogreen assays b Program the PCR Reactions Place the reaction plate in the instrument and set the PCR conditions as follow Cycle Step Temp Time Cycle Activation 98 C 30 sec 1 98 C 10 sec Cycling 55 C 20 sec 22 72 C 20 sec Final Extension 72 C 2 min 1

EpiNext™ High-Sensitivity Bisulfite-Seq Kit

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EpiNext&trade; High-Sensitivity Bisulfite-Seq Kit

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