BisulFlash ™ DNA Modification Kit
Contents
1. BisulFlash DNA Modification Kit Base Catalog P 1026 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The converted DNA obtained with the BisulFlash DNA Modification Kit is suitable for various downstream methylation analyses including conventional MS PCR real time MS PCR MS HRM methylation microarray and bisulfite sequencing including pyrosequencing and deep sequencing Input DNA The amount of DNA for each modification can be 0 2 ng 1 ug For optimal modification the input DNA amount should be 50 200 ng If you use the BisulFlash DNA Modification Kit for MSP with extremely small amounts of starting DNA the number of PCR cycles should be greater than 45 For the best PCR results the target regions to be amplified should be less than 250 bp The yield of DNA purified after bisulfite modification depends on the amount of input DNA nature of DNA and source of the starting material Genomic DNA should be used for bisulfite modification 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 Starting Material Starting materials may include various tissue or cell samples such as cells from flask or microplate cultured cells microdissection sample paraffin embedded tissue plasma serum sample and body fluid sample etc Precautions To a2. DNA solution Note Check 1 if there are precipitates in BF1 solution bottle before adding it to the vial If so shake the bottle to re dissolve it and 2 if DNA volume is large and concentration is lower than 10 ng ul it is recommended to concentrate DNA using Epigentek s DNA Concentrator Kit Cat No P 1006 prior to bisulfite treatment Prepared BF1 BF5 BF6 solution can be stored at room temperature for up to 2 weeks without significant loss of efficiency For the best results the mixed solution should be used immediately 3 Tightly close the PCR tubes and place them in a thermal cycler with heated lid Program and run the thermal cycler at 95 C for 20 min Note If the DNA template contains high GC region or secondary structure the following program for sequencing can be used instead Alternative Enhanced Program for Ideal Sequencing Results Optional 95 C 4 min 65 C 30 min 95 C 4 min 65 C 30 min 95 C 4 min 65 CT 60 min Hold 18 20 T up to 6 h Meanwhile insert the number of F Spin Columns column into F Collection Tubes collection tube as needed by your experiment Converted DNA Clean Up 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 2015 11 16 Epigentek Group Inc All rights reserved Products are for research use only P 1026 Add 250 ul of BF2 to each column Then tr
3. for diagnostic or therapeutic application Intellectual Property The BisulFlash DNA Modification Kit and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW DNA methylation occurs by the covalent addition of a methyl group at the 5 carbon of the cytosine ring resulting in 5 methylcytosine There are various methods used to assess DNA methylation states However only bisulfite modification of genomic DNA followed by PCR amplification cloning and sequencing of individual PCR amplimers yields reliable information on the methylation states of individual cytosines on individual DNA molecules By treating DNA with bisulfite cytosine residues are deaminated to uracil while leaving 5 methylcytosine intact Unmethylated DNA Methylated DNA Original Sequence C C G T C G A C G T C C G T C G A C G T After Bisulfite Conversion U U G T U G A U G T U C G T C G A C G T The traditional bisulfite conversion method needs 12 16 h for bisulfite treatment resulting in heavy DNA degradation gt 80 high inappropriate methylcytosine deamination gt 3 5 and low cytosine conversion rate lt 95 In March 2005 Epigentek became the first company to develop a fast DNA bisulfite modification method to overcome these problems shortening the entire bisulfite process from 16 hours to just 1 5 hours significantly improving cytosine conversion efficiency gt 99 9 and effectively preventing converted DNA d
4. A capture solution enables DNA to tightly bind to the column filter thus DNA cleaning can be carried out on the column to effectively remove residual bisulfite and salts Schematic procedure of the BisulFlash DNA Modification Kit to obtain converted DNA 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 4 Printed 2015 11 16 P 1026 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 200 pg to 1 ug per reaction An optimal amount is 50 200 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 Bisulfite DNA Conversion 1 Addi ml of BF1 to 1 wal of BF5 Mix by inverting and shaking the vial repeatedly for 2 min Add 80 ul of BF6 to the wal and mix by inverting and shaking for an additional 3 min trace amount of undissolved BF5 may remain which is normal as BF5 is saturated in solution 2 For each 0 2 mI PCR tube add 110 ul of the mixed BF1 BF5 BF6 solution followed by adding 1 5 ul of
5. IED O Thermal cycler with heated lid Since the bisulfite reaction is not overlaid wth mineral oil only thermal cyclers wth heated lids are suitable for this procedure Desktop centrifuge up to 14 000 rom Pipette and pipette tips 0 2 ml PCR tubes 1 5 ml microcentrifuge tubes Oo OF o 90 ethanol 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 2 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2015 11 16 Epigentek Group Inc All rights reserved Products are for research use only P 1026 GENERAL PRODUCT INFORMATION Quality Control Each lot of BisulFlash DNA Modification Kits 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 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 Modification Kit is for research use only and is not intended
6. ansfer the samples from each PCR tube from Step 2 to each column containing the BF2 Centrifuge at 12 000 rom for 30 sec Remove columns from collection tubes and discard the flowthrough Place columns back into collection tubes Add 200 ul of 90 ethanol solution to each column Centrifuge at 12 000 rom for 20 sec Prepare final desulphonation buffer by adding 12 ul of BF3 to every 1 ml of 90 ethanol and mix Add 60 ul of the final desulphonation buffer BF3 and 90 ethanol mixture to each column Allow columns to sit for 8 min at room temperature then centrifuge at 12 000 rom for 20 sec Remove columns from collection tubes and discard the flowthrough Place columns back into collection tubes Add 200 ul of 90 ethanol to each column Centrifuge at 12 000 rom for 20 sec Remove columns from collection tubes and discard the flowthrough Place columns back into collection tubes Add 200 ul of 90 ethanol to each column again and centrifuge at 12 000 rom for 30 sec Insert each column into a new 1 5 ml tube Add 10 20 ul of BF4 directly to each column s filter membrane Centrifuge at 12 000 rom 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 and 2 4 ul for each end point PCR Methylation specific real time PCR can be performed by using your own successful method For your convenience and the best results Epi
7. brane of the entire sample has passed through the column filter membrane Poor Results in Little or no PCR producteven in Ensure that all PCR components were Downstream positive control added and that suitable PCR program is Methylation Specific used PCR cycle should be gt 40 110 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 2015 11 16 Epigentek Group Inc All rights reserved Products are for research use only P 1026 PCR primers and probes were not appropriate or were incorrectly designed Ensure the primer and probes are suitable for MS PCR and the target regions to be amplified are less than 250 bps Ensure the amountof template DNA used in PCR was sufficient Significantnon specific PCR Failed bisulfite conversion Ensure that products allsteps of the modification and cleanup protocol were followed and that input DNA amountis within the recommended range Primers and probes are notspecific for converted DNA and target genes Check the primer and probe design RELATED PRODUCTS DNA Sample Preparation 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 C
8. e DNA or too much DNA Increase or decrease input DNA to within i e lt 100 pg or gt 1 ug the correct range or to the optimal amountof 50 200 ng Template contains high GC region Increase the thermal cycler program time or secondarystructure by 5 10 minin Step 3 Temperature or thermal cycling Check for appropriate temperature or condition is incorrect thermal cycling conditions Insufficient DNA clean up Ensure that 12 ul of BF3 is added into every 1 mlof 90 ethanolin Step 6 Solution BF1 contains precipitates Checkif there are precipitates in the bottle of BF1 solution prior to adding it to the tube If so shake the bottle until re dissolved Solution BF1 was contaminated by Checkif BF1 solution has anycolor other chemicals or affected by change deep yellow or brown or long term exposure to air indissoluble precipitates If so use order new BF1 solution Kit is not stored or handled Store all components ofthe kit at room properly temperature Tightly cap the BF1 solution after each opening oruse Eluate Contains Little Poor input DNA quality degraded Checkif DNAis degraded by running gel or No DNA Buffer BF2 Capture Solution is Ensure that BF2 is addedin Step 4 not added into the sample Concentration of ethanol solution Use 90 ethanol for DNA clean up used for DNAclean up is not correct Sample is notcompletely passed Centrifuge for 1 min at 12 000 rpm or until through the filter mem
9. egradation To effectively and efficiently prepare converted DNA for use in various downstream analyses an ideal DNA bisulfite modification method should be 1 highly accurate to allow for the complete conversion of cytosine to uracil correct conversion without deamination of methylcytosine to thymine inappropriate conversion and 2 rapid enough to enable the bisulfite process to be as short as possible as rapid DNA methylation analysis is highly demanded for basic research and particularly for clinical applications Epigentek continues to innovate with the development of the BisulFlash DNA Modification Kit perfecting DNA bisulfite treatment for better DNA methylation analysis This kit greatly improves the currently used methods kits for DNA bisulfite modification With the novel and optimized bisulfite composition the BisulFlash DNA Modification Kit allows for the DNA modification step to be just 20 110 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 Group Inc All rights reserved Products are for research use only P 1026 minutes with a complete cytosine conversion More importantly it greatly reduces inappropriate conversion of 5 methylcytosine to thymine lt 0 1 The BisulFlash DNA Modification Kit is suitable for MS PCR real time MS PCR methylation microarray and bisulfite sequencing
10. ell DNA Extraction Kit DNA Bisulfite Modification P 1002 Methylamp Coupled DNA Isolation amp Modification Kit P 1008 Methylamp 96 DNA Modification Kit P 1016 Methylamp Whole Cell Bisulfite 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 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page g 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 1026
11. gentek offers the Methylamp MS qPCR Fast Kit that is optimized for fast methylation specific qPCR reactions in 70 minutes see Working with Methylation Specific qPCR WORKING WITH METHYLATION SPECIFIC qPCR When working with MS qPCR we recommend using the Methylamp MS qPCR Fast Kit Cat No P 1028 which contains a hot start polymerase system and has been optimized to decrease the overall methylation specific qPCR amplification time The master mix is provided at 2X 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 Companent Veihyamp Waser WAKER Reverse Primer 0 4 0 5 uM DNA Template 50 pg 0 1 ug DNAVRNA free H20 6 7 ul Total Volume For the negative control use DNA RNA free water instead of DNA template Program the PCR Reactions Cycle Step Cyce i 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Pase 6 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2015 11 16 Epigentek Group Inc All rights reserved Products are for research use only P 1026 Cycling Final Extension TROUBLESHOOTING Possible Causes Suggestions DNA is Poorly Modified Poor DNA quality DNA is severely Check if the sample DNA260 280 ratio is degraded between 1 6 1 9 and if DNA is degraded by running gel Too littl
12. including pyrosequencing and deep sequencing The BisulFlash DNA Modification Kit has the following advantages and features e Convenient single temperature incubation without the need for a separate DNA denaturation step e The fastest and most convenient protocol that can be finished in as short as 30 minutes e Completely converts unmethylated cytosine into uracil gt 99 9 with negligible inappropriate or error conversion of methylcytosine to thymine lt 0 1 e Powerful protection against DNA degradation with over 90 of DNA loss prevented e Extremely low requirement of input DNA for modification only 0 2 ng or 50 cells e Simple reliable and consistent modification conditions PRINCIPLE amp PROCEDURE Simultaneous DMA denaturation and modification CUCU DNA Clean up OG 10 Converted DNA Elution As anext generation bisulfite conversion tool the BisulFlash DNA Modification Kit contains all reagents required for an ultra fast bisulfite conversion ona DNA sample With the unique conversion mix solution which contains powerful DNA protection reagents DNA denaturation status is sustained throughout the entire bisulfite DNA conversion process thereby enabling 100 of DNA to be modified in single stranded form without chemical and thermophilic degradation Thus this novel approach leads to an accelerated conversion of all cytosine to uracil with negligible methylcytosine deamination The non toxic DN
13. void cross contamination the following precautions are necessary for handling Epigentek F Spin Columns Carefully pipette the sample or solution into the F Spin Column Use aerosol barrier pipette tips and always change pipette tips between liquid transfers Always cap the F Spin Columns before placing them in a microcentrifuge Wear gloves throughout the entire procedure In case of contact between gloves and sample change gloves immediately 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 1 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2015 11 16 Epigentek Group Inc All rights reserved Products are for research use only P 1026 KIT CONTENTS BF1 Conversion Mix Solution BF2 Capture Solution BF3 Desulphonation Solution 60 ul BF4 Elution Solution BF5 Conversion Enhancer BF6 Denaturation Enhancer 600 ul F Spin Column F Collection Tube User Guide Always cap spin columns before placing them in the microcentrifuge SHIPPING amp STORAGE The kit is shipped at ambient temperature Store all components at room temperature 15 22 C away from light The kit can be stable for up to 6 months from the shipment date when stored properly Note Check if there are any precipitates in the bottle of BF1 solution prior to use If so shake the bottle to re dissolve it Tightly cap BF1 after each opening or usage MATERIALS REQUIRED BUT NOT SUPPL
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