BisulFlash™ DNA Bisulfite Conversion Mag-96 Kit
Contents
1. BisulFlash DNA Bisulfite Conversion Mag 96 Kit Base Catalog P 1050 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The BisulFlash DNA Bisulfite Conversion Mag 96 Kit uses a unique conversion solution and magnetic binding bead clean up to generate bisulfite converted DNA in a fast reliable and high throughput format Each kit contains sufficient components for 96 total reactions Input DNA The amount of DNA for each reaction can be 0 1 1 ug For an optimal reaction the input DNA amount should be 100 ng If small amounts ex lt 10 ng of starting DNA are used the number of PCR cycles should be greater than 45 The yield of purified DNA after bisulfite modification depends on the amount of input DNA nature of DNA and source of the starting material Starting Material Starting materials may include various tissue or cell samples such as cultured cells from a flask or microplate microdissection sample paraffin embedded tissue plasma serum sample and body fluid sample etc Precautions To avoid cross contamination the following precautions are necessary for handling samples in tube vials 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 Tel2. 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2015 11 16 Epigentek Group Inc All rights reserved Products are for research use only P 1050 KIT CONTENTS Component 96 Reactions 192 reactions Storage Cat P 1050 096 Cat P 1050 192 Upon Receipt BisulFlash Conversion Mix 4 ml 8ml RT DNA Binding Solution 15 ml 30 ml RT Desulfonation Buffer 0 6 ml 1 2 ml RT Elution Buffer 2 ml 4 ml RT Binding Beads 0 5 ml 1ml 4 C Mag 96 Well Plate 1 2 RT Adhesive Cover Film 1 2 RT User Guide 1 1 RT SHIPPING amp STORAGE The kit is shipped at ambient room temperature Upon receipt Store Binding Beads at 4 C Store other components at room temperature 15 22 C away from light All components of the kit are stable for up to 6 months from the date of shipment when stored properly MATERIALS REQUIRED BUT NOT SUPPLIED O Thermal cycler with heated lid Since the bisulfite reaction is not overlaid with mineral oil only thermal cyclers with heated lids are suitable for this procedure Pipette and pipette tips 0 2 ml PCR tubes and PCR plate Magnetic stand 96 well format 1 5 ml microcentrifuge tubes 0 0 0 0 O 90 ethanol GENERAL PRODUCT INFORMATION Quality Control Each lot of BisulFlash DNA Bisulfite Conversion Mag 96 Kit is tested against predetermined specifications to ensure consistent product quality Epigentek guarantees
3. Aberrant DNA methylation is associated with pathogenesis of diseases such as cancer autoimmune disorders and schizophrenia Thus gene region specific or genome wide analysis of DNA methylation or 5 methylcytosine 5 mC could provide valuable information for discovering epigenetic markers used for disease diagnosis and potential targets used for therapeutics Bisulfite modification of genomic DNA followed by PCR amplification cloning sequencing or whole genome sequencing is currently considered to be the most reliable method in assessing the methylation states of individual cytosine on individual DNA molecules By treating DNA with bisulfite cytosine residues are deaminated to uracil while leaving 5 methylcytosine intact Sulfonation Deamination Desulfonation NH2 H2 NI HSO 3 3 oo Dur NH cytosine cytosine SO uracil SO uracil ry a 5 methylcytosine residues are left Ay OH unchanged during the bisulfite H modification process 5 methylcytosine However the currently used DNA bisulfite conversion methods are still lacking especially in speed convenience and throughput To address this issue Epigentek continues to innovate with the development of the BisulFlash DNA Bisulfite Conversion Mag 96 Kit With novel procedures and 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 11 16 Epigentek Grou
4. Kit Page 8 Printed 2015 11 16 P 1050 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 Modification P 1001 Methylamp DNA Modification Kit P 1016 Methylamp Whole Cell Bisulfite Modification Kit P 1026 BisulFlash DNA Modification Kit DNA Methylation Analysis P 1005 TuMinute PCR Clean Up Kit P 1011 Methylamp Universal Methylated DNA Kit P 1019 Methylamp Universal Methylated DNA Preparation Kit P 1028 Methylamp MS gPCR Fast Kit Magnetic Devices O10002 EpiMag HT 96 Well Magnetic Separator 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 2015 11 16 Epigentek Group Inc All rights reserved Products are for research use only P 1050
5. long term exposure to air Check if BisulFlash Conversion Mix solution has any color change deep yellow or brown or insoluble precipitates If so use order new BisulFlash Conversion Mix solution Kit is not stored or handled properly Store all components of the kit at room temperature except Binding Beads Tightly cap the BisulFlash Conversion Mix solution after each opening or use 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 11 16 P 1050 Eluate Contains Little or No DNA Poor input DNA quality degraded Check if DNA is degraded by running gel DNA Binding Solution is not sufficiently added into the sample Ensure that DNA Binding Solution is added in Step 1 of Converted DNA Clean Up Concentration of ethanol solution used for DNA clean up is not correct Use 90 ethanol for DNA clean up DNA is not completely bound to the beads at the DNA binding step or beads are overdried at the DNA elution step Make sure the incubation time is sufficient 10 min at Step 1 of Converted DNA Clean Up and or the beads are not over dried at Step 8 of Converted DNA Clean Up Poor Results in Downstream Methylation Specific PCR Little or no PCR product even in posit
6. thereby enabling 100 of unmethylated cytosine to be converted to uracil Desulphonation and clean up of the converted DNA is performed using magnetic bead based binding and separation in a 96 well microplate High yield converted DNA can be obtained and used for various downstream applications including PCR array and next generation sequencing DNA denature and 104 2 modification 10 1 DNA clean up 10 0 10 1 0 2 4 6 8 101214 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 Converted Cycle DNA elution Fig 2 Different amounts of human genomic DNA were converted using the BisulFlash DNA Bisulfite Conversion Mag 96 Kit Converted DNA Fig 1 Schematic procedure of the was amplified using Methylamp MS gPCR Fast BisulFlash DNA Bisulfite Conversion Kit Cat No P 1028 Mag 96 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 2015 11 16 Epigentek Group Inc All rights reserved Products are for research use only P 1050 Original Unconverted Sequence C CAGG C CCCG CCG CTG Bisulfite Converted Sequence T TAGG TTTCGTCGTTG Fig 3 High accuracy of DNA conversion is achieved by the BisulFlash DNA Bisulfite Conversion Mag 96 Kit 100 ng of genomic DNA methylated in all CpG sites by DNA methylases was treated with the kit followed by real time qPCR amplification using primers for multiple promoters containing
7. www epigentek com Printed 2015 11 16 Epigentek Group Inc All rights reserved Products are for research use only P 1050 Converted DNA Clean Up 1 Add 120 ul of DNA Binding Solution to each PCR well tube followed by adding 5 ul of Binding Beads mix by pipetting 8 10 times and then transfer the mixed samples from each PCR well tube to the wells of the Mag 96 well plate Incubate at RT for 10 min Note The un used plate wells can be covered with the Adhesive Covering Film include in the kit Put the plate on an EpiMag HT Magnetic Separator or an appropriate magnetic stand until the solution is clear about 2 minutes Carefully remove and discard the supernatant Caution Be careful not to disturb or discard the beads that contain DNA Add 200 ul of 90 ethanol solution to each well and resuspend the beads Place the plate on the magnet stand for 2 minutes or until the solution is clear Remove and discard supernatant Repeat Step 3 once for a total of 2 washes Prepare final desulphonation buffer by adding 25 ul of Desulfonation Buffer to every 1 ml of 90 ethanol and mix Add 200 ul of the final desulphonation buffer to each well and resuspend beads Incubate at room temperature for 15 min and then put the plate on the magnet stand for 2 minutes or until the solution is clear Remove and discard the supernatant Add 200 ul of 90 ethanol solution to each well and resuspend the beads Place the plate on the magnet stand for
8. 2 minutes or until the solution is clear Remove and discard supernatant Repeat Step 6 once for a total of 2 washes Make sure that the ethanol is completely removed after the final wash Air dry beads for 3 4 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 Converted DNA Elution 1 Resuspend the beads in 20 ul 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 19 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 WORKING WITH METHYLATION SPECIFIC qPCR Working with MS gPCR not only allows detection of gene specific methylation it also validates if the DNA is efficiently converted For MS qPCR we recommend using the Methylamp MS gPCR Fast Kit Cat No P 1028 which has been optimized to decrease the overall methylation specific gPCR amplification time and include primers specific for converted DNA The master mix is provided at 2X 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 epigen
9. ive control Ensure that all PCR components were added and that a suitable PCR program is used PCR cycles should be gt 40 PCR primers and probes were not appropriate or were incorrectly designed Ensure the primer and probes are suitable for MS PCR Ensure the amount of template DNA used for PCR was sufficient Insufficient DNA clean up Ensure that 25 ul of Desulfonation Buffer is added into every 1 ml of 90 ethanol at Step 5 of Converted DNA Clean Up Beads were not sufficiently re suspended at the wash and desulfonation steps Ensure that the beads have been completely re suspended during the wash and desulfonation steps Significant non specific PCR products Failed bisulfite conversion Ensure that all steps of the modification and clean up protocol were followed and that the input DNA amount is within the recommended range Primers and probes are not specific for converted DNA and target genes Check the primer and probe design RELATED PRODUCTS DNA Sample Preparation P 1003 P 1004 P 1006 P 1007 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 M General Tissue Section DNA Isolation Kit FitAmp Plasma Serum DNA Isolation Kit DNA Concentrator Kit FitAmp M Gel DNA Isolation
10. numerous CpG sites and then directly sequenced 100 C at non CpG sites are converted to T and 100 C at CpG sites remain as C ASSAY PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Input DNA Amount Optimal DNA amount is 100 ng per reaction Starting DNA may be in water or in a buffer such as TE 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 For the magnetic stand used for capturing Binding Beads we recommend using Epigentek s EpiMag HT Magnetic Separator Cat No Q10002 which has very strong magnetic power and is proven to quickly and efficiently achieve high reproducible retention of magnetic bead bound DNA in various 96 well plates and specifically suitable for the Mag 96 well plate included in this kit Bisulfite DNA Conversion 1 For each well of a PCR plate or 0 2 ml PCR tube add 30 ul of the BisulFlash Conversion Mix solution followed by adding 1 4 ul of your DNA sample optimal 100 ng 2 Tightly close the PCR well tubes and place them in a thermal cycler with heated lid Program and run the thermal cycler at 80 C for 45 min 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
11. p Inc All rights reserved Products are for research use only P 1050 optimized components the kit allows for preparing bisulfite converted DNA in a high throughout format within 1 h and 20 min The kit has the following advantages and features e Fast Complete the entire procedure in as little as 1 hour and 20 min for as many as 96 samples simultaneously averaging just 50 seconds per sample e Convenient Ready to use liquid conversion mix is simply added to the DNA samples directly without need for pre preparation of the conversion reagent e Streamlined Concurrently processes the DNA denaturation and C to T conversion steps without the need for a separate DNA denaturation step e Complete Conversion Completely converts unmethylated cytosine into uracil gt 99 9 with negligible inappropriate or error conversion of methylcytosine to thymine lt 0 1 e Flexible Choice of either a manual with one single reaction each time or b high throughput with 96 reactions each time can be used making the assay flexible e Robust Simple reliable and consistent reaction conditions with an easy to follow protocol and high yield PRINCIPLE amp PROCEDURE The BisulFlash DNA Bisulfite Conversion Mag 96 Kit contains all reagents required for a fast bisulfite conversion in a high throughput format With the unique conversion mix solution DNA denaturation status is sustained throughout the entire bisulfite conversion process
12. tek com Printed 2015 11 16 Epigentek Group Inc All rights reserved Products are for research use only P 1050 concentration for easier preparation of PCR reactions requiring only the addition of primers and templates With this kit the MS qPCR can be finished in as short as 70 min Prepare the PCR Reactions Component Size pl Final Concentration Methylamp Master Mix 2X 10 ul 1X Forward Primer 1 ul 0 4 0 5 uM Reverse Primer 1 ul 0 4 0 5 uM DNA Template 1 2 ul 50 pg 0 1 ug DNA RNA free H O 6 7 ul Total Volume 20 ul For the negative control use DNA RNA free water instead of DNA template Program the PCR Reactions Cycle Step Temp Time Cycle Activation 95 C 7 min dl 95 C 10 sec Cycling 55 C 10 sec 40 45 72 C 8 sec Final Extension 72 C 1 min 1 TROUBLESHOOTING Problem Possible Causes Suggestions DNA is Poorly Poor DNA quality DNA is Check if the sample DNA 260 280 ratio Modified severely degraded is between 1 6 1 9 and if DNA is degraded by running gel Too little DNA or too much DNA i e lt 100 pg or gt 1 ug Increase or decrease input DNA to within the correct range or to the optimal amount of 100 ng Temperature or thermal cycling condition is incorrect Check for appropriate temperature or thermal cycling conditions BisulFlash Conversion Mix solution was contaminated by other chemicals or affected by
13. the performance of all products in the manner described in our product instructions 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 2015 11 16 P 1050 Product Warranty If this product does not meet your expectations simply call 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 Usage Limitation The BisulFlash DNA Bisulfite Conversion Mag 96 Kit is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The BisulFlash DNA Bisulfite Conversion Mag 96 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
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