BisulFlash™ DNA Bisulfite Conversion Easy Kit
Contents
1. BisulFlash DNA Bisulfite Conversion Easy Kit Base Catalog P 1054 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The BisulFlash DNA Bisulfite Conversion Easy Kit uses a unique conversion solution and column based clean up to generate bisulfite converted DNA in a fast reliable and convenient format Each kit contains sufficient components for 50 total reactions High yield converted DNA can be obtained and used for various downstream applications including PCR array and next generation sequencing after post bisulfite adaptor ligation 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 i e 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 liguid transfers Wear gloves througho2. RELATED PRODUCTS BisulFlash Conversion Mix solution was contaminated by other chemicals or affected by long term exposure to air Kit is not stored or handled properly Poor input DNA quality degraded DNA Binding Solution is not sufficiently added into the sample Concentration of ethanol solution used for DNA clean up is not correct Little or no PCR product even in positive control Insufficient DNA clean up Significant non specific PCR products DNA Sample Preparation P 1003 P 1004 P 1054110 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 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 Store all components of the kit at room temperature Tightly cap the BisulFlash Conversion Mix solution after each opening or use Check if DNA is degraded by running a gel Ensure that DNA Binding Solution is added in Step 1 of Converted DNA Clean Up Use 90 ethanol for DNA clean up 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 sui
3. sample v 45 min j 1 10 Amount of starting DNA ng Converted DNA clean up B 4 15 min Cycle threshold Ct Cycle Threshold Ct Ready to use converted DNA Gene 1 Gene 2 Gene 3 Gene 4 B actin Fig 1 Schematic procedure of the BisulFlash Fig 2 Different amounts of HeLa DNA were converted using the DNA Bisulfite Conversion Easy Kit BisulFlash DNA Bisulfite Conversion Easy Kit A Real time PCR Ct value curve for different amounts of inout DNA performed using primers designed for both methylated and unmethylated alleles of B actin B Ct values for different gene targets of a converted DNA sample Internal control B actin Ct value is also shown Allconverted DNA w as amplified using the Methylamp MS qPCR Fast Kit Cat No P 1028 P 1054110 Bi County Blvd Ste 122 Farmingdale NY 11735 Pag e4 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 1054 ASSAY PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Inout 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 Stora
4. ack into Collection Tubes Add 200 ul of 9096 ethanol to each Spin Column again and centrifuge at 12 000 rom for 45 sec Insert each column into a new 1 5 ml tube Add 10 20 ul of Elution Buffer directly to each Spin Column s filter membrane Centrifuge at 12 000 rpm for 30 sec to elute converted DNA Modified DNA is now ready for use or storage at or below 20 C for up to 6 months We recommend using 1 2 ul of the DNA for each real time qPCR reaction and 2 4 ul for each end point PCR reaction Methylation specific real time PCR can be performed by using your own successful method For your convenience and the best results Epigentek offers the Methylamp MS qPCR Fast Kit Cat No P 1028 that is optimized for fast methylation specific qPCR reactions in 70 minutes see Work ing with Methylation Specific GPCR P 1054110 Bi County Blvd Ste 122 Farmingdale NY 11735 Pag e5 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 P1054 WORKING WITH METHYLATION SPECIFIC qPCR Working with MS gPCR not only allows detection of gene specific methylation it also validates ifthe DNA is efficiently converted For MS gPCR we recommend using the Methylamp MS qPCR Fast Kit Cat No P 1028 which has been optimized to decrease the overall methylation specific gPCR amplification time and include p
5. ge Isolated genomic DNA can be stored at 4 C or 20 C until use Bisulfite DNA Conversion 1 For each well of a PCR plate or 0 2 ml PCR tube add 150 ul of the BisulFlash Conversion Mix solution followed by adding 2 15 ul of your DNA sample optimal 100 ng 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 Converted DNA Clean Up 1 Place a Spin Column into a 2 ml Collection Tube Add 300 ul of DNA Binding Solution to each Spin Column Then transfer the samples from each PCR tube from Step 2 of Bisulfite DNA Conversion to the Spin Column Centrifuge at 12 000 rom for 30 sec Remove Spin Columns from Collection Tubes and discard the flowthrough Place Spin Columns back into Collection Tubes Add 200 ul of 90 ethanol solution to each Spin Column Centrifuge at 12 000 rom for 30 sec Prepare final desulfonation buffer by adding 25 ul of Desulfonation Buffer to every 1 ml of 90 ethanol and mix Add 150 ul of the final desulfonation buffer to each column Allow Spin Columns to sit for 10 min at room temperature then centrifuge at 12 000 rom for 30 sec Remove Spin Columns from Collection Tubes and discard the flowthrough Place Spin Columns back into Collection Tubes Add 200 ul of 90 ethanol to each Spin Column Centrifuge at 12 000 rom for 30 sec Remove Spin Columns from Collection Tubes and discard the flowthrough Place Spin Columns b
6. in our product instructions P 1054110 Bi County Blvd Ste 122 Farmngdale 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 Epigentek Group Inc All rights reserved Products are for research use only Printed 2015 11 16 P 1054 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 Easy Kit is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The BisulFlash DNA Bisulfite Conversion Easy 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 requlating qene expression in nearly all biological processes including development growth and differentiation Aberrant DNA methylation is associated with pathogenes
7. is 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 NH3 H gt o NH cytosine cytosine SO uracil SO uracil on lt gt 5 methylcytosine residues are left A OH unchanged during the bisulfite H modification process 5 methylcytosine Epigentek developed the BisulFlash DNA Modification Kit Cat No P 1026 to improve DNA bisulfite treatment for faster DNA methylation analysis Epigentek continues to innovate with the development of the BisulFlash DNA Bisulfite Conversion Easy Kit With novel procedures and optimized P 1054110 Bi County Blvd Ste 122 Farmngdale NY 11735 Pag e3 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 resea
8. rch use only P1054 components the kit allows for the preparation of bisulfite converted DNA within 1 hour by simply adding a ready to use liquid conversion mix to the DNA samples directly without need for pre preparation of the conversion reagent The kit also has the following advantages and features e Fast Procedure The entire process can be completed within 1 hour 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 erroneous conversion of methylcytosine to thymine lt 0 1 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 Easy 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 thereby enabling 100 of unmethylated cytosine to be converted to uracil Desulfonation and clean up of the converted DNA is performed using DNA purification columns High yield converted DNA can be obtained and used for various downstream applications including PCR array and next generation sequencing BisulFlash Conversion Mix plus DNA
9. rimers specific for converted DNA The master mix is provided at 2X concentration for easier preparation of PCR reactions reguiring only the addition of primers and templates With this kit the MS gPCR can be finished in as short as 70 min Prepare the PCR Reactions Component Methylamp Master Mix OX Forward Primer Reverse Primer DNA Template er DNA RNA free HO 6 7 ul Total Volume aw OO For the negative control use DNA RNA free water instead of DNA template Program the PCR Reactions Cycle Step 95 C 10 sec Cycling 55 C 10 sec 40 45 72 C 8 sec Final Extension TROUBLESHOOTING 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 a gel Too little DNA or too much DNA Increase or decrease input DNA to i e lt 100 pg or gt 1 ug within the correct range or to the optimal amount of 100 ng Temperature or thermal cycling Check for appropriate temperature or condition is incorrect thermal cycling conditions P 1054110 Bi County Blvd Ste 122 Farmingdale NY 11735 Pag e6 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 1054 Eluate Contains Little or No DNA Poor Results in Downstream Methylation Specific PCR
10. table for MS PCR Ensure the amount of template DNA used for PCR was sufficient Ensure that 25 ul of Desulfonation Buffer is added into every 1 ml of 90 ethanol at Step 3 of Converted DNA Clean Up 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 FitAmp General Tissue Section DNA Isolation Kit FitAmp Plasma Serum DNA Isolation Kit Page 7 Printed 2015 11 16 P 1054 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 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 qPCR Fast Kit Magnetic Devices Q10002 EpiMag HT 96 Well Magnetic Separator P 1054110 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 epigen
11. tek com Printed 2015 11 16 Epigentek Group Inc All rights reserved Products are for research use only P 1054
12. ut the entire procedure In case of contact between gloves and sample change gloves immediately P 1054110 Bi County Blvd Ste 122 Farmngdale NY 11735 Pag el 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 P1054 KIT CONTENTS Cat P 1054 050 Upon Receipt BisulFlash Conversion Mix DNA Binding Solution Desulfonation Buffer Collection Tube Elution Buffer Always cap spin columns before placing them in the microcentrifuge SHIPPING amp STORAGE The kit is shipped at ambient room temperature Upon receipt Store all 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 Ll Oo OF OU Thermal cycler with heated lid Since the bisulfite reaction is not overlaid with mineral oil only thermal cyclers wth heated lids are suitable for this procedure Pipette and pipette tips 0 2 ml PCR tubes or PCR plate 1 5 ml microcentrifuge tubes 9095 ethanol GENERAL PRODUCT INFORMATION Quality Control Each lot of the BisulFlash DNA Bisulfite Conversion Easy Kit is tested against predetermined specifications to ensure consistent product quality Epigentek guarantees the performance of all products in the manner described
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