5-fC - Epigentek
Contents
1. Wash wells then add capture antibody Wash wells then add detection antibody and enhancer solution Add color developing solution for color develop ment then measure absorbance Schematic procedure of the MethylFlash 5 Formylcytosine 5 fC DNA Quantification Kit Colorimetric 0 6 0 5 0 4 0 3 0OD450 nm 0 2 0 1 0 0 1 0 2 0 3 0 4 5 fC ng 5 fC standard control was added into the assay wells at different concentrations and then measured with the MethylFlash 5 Formylcytosine 5 fC DNA Quantification Kit Colorimetric 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 08 20 Epigentek Group Inc All rights reserved Products are for research use only P 1041 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 100 ng to 300 ng per reaction An optimal amount is 300 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 See Ordering Information DNA Storage Isolated genomic DNA can be stored at 4 C short term or 20 C long term until use 1 Buffer and Solution Preparation a Prepar2. MethylFlash 5 Formylcytosine 5 fC DNA Quantification Kit Colorimetric Base Catalog P 1041 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The MethylFlash 5 Formylcytosine 5 fC DNA Quantification Kit Colorimetric is a complete set of optimized buffers and reagents to colorimetrically quantify 5 formylcytosine 5 fC in a microplate based format It is suitable for use with DNA isolated from any species mammals plants fungi bacteria viruses etc in a variety of forms including but not limited to cultured cells fresh and frozen tissues paraffin embedded tissues and body fluid samples Input DNA The amount of DNA for each assay can be 100 ng to 300 ng For optimal quantification the input DNA amount should be 300 ng as 5 fC content is generally less than 0 01 of total DNA Starting Material Starting materials can include various tissue or cell samples for example cells from flask or microplate cultured cells fresh and frozen tissues paraffin embedded tissues blood body fluid samples etc Internal Control Both negative and positive DNA controls are provided in this kit A standard curve can be performed range 10 pg to 400 pg of 5 fC or a single quantity of 5 fC can be used as a positive control Because 5 fC content can vary from tissue to tissue and from normal and diseased states or vary under treated and untreated conditions it is advised to run replicate samples to ensure that the signal generated
3. Colorimetric 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 MethylFlash 5 Formylcytosine 5 fC DNA Quantification Kit Colorimetric is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The MethylFlash 5 Formylcytosine 5 fC DNA Quantification Kit Colorimetric 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 by DNA methyltransferases resulting in 5 methylcytosine 5mC In somatic cells 5mC is found almost exclusively in the
4. W__1__________ x 100 0 007 0 320 0 120 0 2 Absolute Quantification To quantify the absolute amount of 5 fC using an accurate calculation first generate a standard curve and plot the OD values versus the amount of PC at each concentration point Next determine the slope OD ng of the standard curve using linear regression Microsoft Excel s linear regression functions are suitable for such calculation and the most linear part at least 4 concentration points including 0 point of the standard curve for optimal slope calculation Now calculate the amount and percentage of 5 fC in your total DNA using the following formulas Sample OD NC OD 5 fC Amount ng S fC ng me 5 fC h x 100 Slope S S is the amount of input sample DNA in ng 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 Epigentek Group Inc All rights reserved Products are for research use only Printed 2015 08 20 P 1041 Example calculation Average OD450 of NC is 0 120 Average OD450 of sample is 0 141 Slope is 1 OD ng S is 300 ng 0 141 0 120 5 fC ng __ 0 021 ng k 0 021 5 f C _ X 100 0 007 300 SUGGESTED STRIP WELL SETUP Table 1 Single Point Positive Control The suggested strip well plate setup using a single point positive control in a 48 assay format in a 96 assay format Strips
5. is validated This kit will allow the user to quantify an absolute amount of 5 fC and determine the relative 5 fC states of two different DNA samples Precautions To avoid cross contamination carefully pipette the sample or solution into the strip wells 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 2015 08 20 Epigentek Group Inc All rights reserved Products are for research use only P 1041 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 1041 48 Cat P 1041 96 Upon Receipt WB 10X Wash Buffer 14 ml 28 ml 4 C BS Binding Solution 5ml 10 ml RT NC Negative Control 10 ug ml 10 ul 20 ul 20 C PC Positive Control 5 fC 1 ug ml 10 ul 20 ul 20 C CA Capture Antibody 1000X 4ul 8 ul 4 C DA Detection Antibody 2000X 6 ul 12 ul 20 C ES Enhancer Solution 5 ul 10 ul 20 C DS Developer Solution 5 ml 10 ml 4 C SS Stop Solution 5 ml 10 ml RT 8 Well Assay Strips With Frame 6 12 4 C User Guide 1 1 RT Spin the solution down to the bottom prior to use Note The NC Negative Control is an oligo containing no 5 fC The P
6. 7 to 12 can be configured as Sample The controls and samples can be measured in duplicate as Strip 1 Strip 2 Strip 3 Strip 4 Strip 5 Strip 6 A NC NC Sample Sample Sample Sample B PC PC Sample Sample Sample Sample c Sample Sample Sample Sample Sample Sample D Sample Sample Sample Sample Sample Sample E Sample Sample Sample Sample Sample Sample F Sample Sample Sample Sample Sample Sample G Sample Sample Sample Sample Sample Sample H Sample Sample Sample Sample Sample Sample Table 2 Standard Curve Preparation The suggested strip well plate setup for standard curve preparation in a 48 assay format in a 96 assay format Strips 7 to 12 can be configured as Sample The controls and samples can be measured in duplicate Well Strip1 Strip 2 Strip3 Strip4 Strip5 Strip6 A NC NC Sample Sample Sample Sample B PC 10 pg ul PC 10 pg ul Sample Sample Sample Sample c PC 20 pg ul PC 20 pg ul Sample Sample Sample Sample D PC 40 pg ul PC 40 pg ul Sample Sample Sample Sample E PC 100 pg ul PC 100 pg ul Sample Sample Sample Sample F PC 200 pg ul PC 200 pg ul Sample Sample Sample Sample G PC 400 pg ul PC 400 pg ul Sample Sample Sample Sample H Sample Sample Sample Sample Sample Sample Suggested Working Buffer and Solution Setup Table 3 Approximate amount of required buffers and solutions for defined assay wells based on the proto
7. C Positive Control is a 5 fC oligo and is normalized to have 100 of 5 fC SHIPPING amp STORAGE The kit is shipped in two parts the first part at ambient room temperature and the second part on frozen ice packs at 4 C Upon receipt 1 Store NC PC DA and ES at 20 C away from light 2 Store WB CA DS and 8 Well Assay Strips at 4 C away from light 3 Store remaining components BS and SS at room temperature away from light Note Check if the wash buffer WB contains salt precipitates before using If so warm at room temperature or 37 C and shake the buffer until the salts are re dissolved All components of the kit are stable for 6 months from the date of shipment when stored properly MATERIALS REQUIRED BUT NOT SUPPLIED O Adjustable pipette Aerosol resistant pipette tips Microplate reader capable of reading absorbance at 450 nm oO m O 1 5 ml microcentrifuge tubes O Incubator for 37 C incubation oO Plate seal or Parafilm M 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 08 20 P 1041 O Distilled water O 1XTE buffer pH 7 5 to 8 0 Oo Isolated DNA of interest GENERAL PRODUCT INFORMATION Quality Control Each lot of the MethylFlash 5 Formylcytosine 5 fC DNA Quantification Kit
8. 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 08 20 Epigentek Group Inc All rights reserved Products are for research use only P 1041 NH NH NH NH NH N nA CH nA i a nA rr DNMT TET1 2 3 TET1 2 3 TET1 2 3 eee ATN 7 Fe ATP Fe ATP N SAM SAH N aKG O co N aKG O co N aKG O co O N H H H H H Cytosine 5 methylcytosine 5 hydroxymethylcytosine 5 formylcytosine 5 carboxycytosine 5mC 5hmC 5fC 5CaC Active demethylation of 5 methylcytosine by TET mediated oxidation to 5 hydroxymethylcytosine 5 formylcytosine and 5 carboxylcytosine followed by decarboxylation base excision repair Wu et al Nat Rev Mol Cell Biol 11 607 620 2010 Several chromatography based techniques such as HPLC MS are used for the detection of 5 fC in tissue and cells However these methods are time consuming require large amounts of DNA and have low throughput with high costs To address these problems Epigentek offers the MethylFlash 5 Formylcytosine 5 fC DNA Quantification Kit Colorimetric which uses a unique microplate based procedure to directly quantify 5 fC The kit has the following advantages and features e Colorimetric assay with easy to follow steps for convenience and speed The entire procedure can be completed within 3 hours and 45 minutes High sensitivity of which the detection limit can be as low as 1 pg o
9. ature are incorrect Ensure the incubation time and temperature described in the protocol are followed correctly Insufficient input materials Ensure that a sufficient amount of positive control gt 50 pg and samples 300 ng is added into the wells Incorrect absorbance reading Check if appropriate absorbance wavelength 450 nm is used Kit was not stored or handled properly Ensure all components of the kit were stored at the appropriate temperature and the cap is tightly capped after each opening or use No signal or weak signal in only the positive control wells The positive control is insufficiently added to the well in Step 2c Ensure a sufficient amount of positive control DNA is added The PC Positive Control is degraded due to improper storage conditions Follow the Shipping amp Storage guidance of this User Guide for storage of PC Positive Control 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 10 Printed 2015 08 20 P 1041 High background present in the negative control wells Insufficient washing of wells Check if washing recommendations at each step are performed according to the protocol Contaminated by sample or positive control Ensure the wel
10. col 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 08 20 Epigentek Group Inc All rights reserved Products are for research use only P 1041 Reagents 1 well 8 wells 16 wells 48 wells 96 wells 1 strip 2 strips 6 strips 12 strips Diluted WB 2 5 ml 20 ml 40 ml 120 ml 240 ml BS 80 ul 640 ul 1300 ul 3900 ul 8000 ul Diluted CA 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted DA 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted ES 50 ul 400 ul 800 ul 2400 ul 4800 ul DS 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml SS 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml NC N A 0 5 ul 1l 0 5 ul 2ul iul 4u J2ul 8ul PC N A 0 5 ul 1 ul 0 5 ul 2yul iul 4u 2 ul 8 pul TROUBLESHOOTING Problem Possible Cause Suggestion No signal in both the positive control and sample wells Reagents are added incorrectly Check if reagents are added in the proper order and if any steps in the protocol may have been omitted by mistake The well is incorrectly washed before DNA binding Ensure the well is not washed prior to adding the positive control and sample The bottom of the well is not completely covered by the BS Binding Solution Ensure the solution coats the bottom of the well by gently tilting from side to side or shaking the plate several times Incubation time and temper
11. context of paired symmetrical methylation of the dinucleotide CpG whereas in embryonic stem ES cells a substantial amount of 5mC is also observed in non CpG contexts The biological importance of 5mC as a major epigenetic modifier of phenotype and gene expression has been recognized widely 5 hydroxymethylcytosine 5hmC as a sixth DNA base with functions in transcription regulation has been detected to be abundant in the human brain the mouse brain and in embryonic stem ES cells In mammals it can be generated by the oxidation of 5mC a reaction mediated by the ten eleven translocation TET family of 5mC hydroxylases 5hmC can be further oxidized to 5 formylcytosine 5 fC and 5 carboxylcytosine 5 caC through TET hydroxylases 5 formylcytosine 5 fC has recently been found in mammalian tissue and cells and has been classified as a seventh DNA base The function of 5 fC in gene regulation is not yet entirely clear but it has been shown that 5 fC exhibits replication dependent dilution during mouse pre implantation development and could be functional in regulating pre implantation development as a whole The detection of 5 fC in various tissues and cells is important because 5 fC could be a marker to indicate active DNA demethylation 5 fC can also be directly excised by Thymine DNA glycosylase TDG to allow subsequent base excision repair BER processing which converts modified cytosine back to its unmodified state 110 Bi County Blvd
12. dale 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 Printed 2015 08 20 Epigentek Group Inc All rights reserved Products are for research use only P 1041 Resulting PC Tube PC 400 pgp IXTE concentration 1 1 0 ul 39 0 ul 10 pg ul 2 1 0 ul 19 0 ul 20 pg ul 3 1 0 ul 9 0 ul 40 pg ul 4 1 0 ul 3 0 ul 100 pg ul 5 2 0 ul 2 0 ul 200 pg ul 6 3 0 ul 0 0 ul 400 pg ul Note Keep each of the diluted solutions except Diluted WB 1X Wash Buffer on ice until use Any remaining diluted solutions other than Diluted WB should be discarded if not used within the same day 2 DNA Binding a Predetermine the number of strip wells required for your experiment Carefully remove un needed strip wells from the plate frame and place them back in the bag seal the bag tightly and store at 4 C b Add 80 ul of BS Binding Solution to each well c Add 1 ulof NC 1 ul of Diluted PC see note below and 300 ng of your Sample DNA 1 8 ul into the designated wells depicted in Table 1 or Table 2 Mix solution by gently tilting from side to side or shaking the plate several times Ensure the solution coats the bottom of the well evenly Note 1 For a single point control add 1 ul of PC at a concentration of 200 pg ul as prepared in Step 1e For the standard curve add 1 ul of Diluted PC at concentrations of 10 to 400 pg ul see the chart in Ste
13. e Diluted WB 1X Wash Buffer 48 Assay Kit Add 13 ml of WB 10X Wash Buffer to 117 ml of distilled water final pH 7 2 7 5 96 Assay Kit Add 26 ml of WB 10X Wash Buffer to 234 ml of distilled water final pH 7 2 7 5 This Diluted WB 1X Wash Buffer can now be stored at 4 C for up to six months b Prepare Diluted CA Capture Antibody Solution Dilute CA Capture Antibody with Diluted WB at a ratio of 1 1000 i e add 1 ul of CA to 1000 ul of Diluted WB About 50 ul of this Diluted CA solution will be required for each assay well c Prepare Diluted DA Detection Antibody Solution Dilute DA Detection Antibody with Diluted WB at a ratio of 1 2000 i e add 1 ul of DA to 2000 ul of Diluted WB About 50 ul of this Diluted DA solution will be required for each assay well d Prepare Diluted ES Enhancer Solution Dilute ES Enhancer Solution with Diluted WB at a ratio of 1 5000 i e add 1 ul of ES to 5000 ul of Diluted WB About 50 ul of this Diluted ES solution will be required for each assay well e Prepare Diluted PC Positive Control Single Point Control Prep Dilute PC Positive Control with 1X TE to 200 pg ul 2 pl PC 8 ul TE Suggested Standard Curve Prep First dilute PC to 400 pg l 4 ul of PC 6 ul of 1X TE Then further prepare 6 different concentrations with the 400 pg ul diluted PC and 1X TE into 10 20 40 100 200 and 400 pg ul according to the following dilution chart 110 Bi County Blvd Ste 122 Farming
14. f 5 fC High specificity with detection of only 5 fC No cross reactivity to cytosine 5 mC 5 hmC and 5 caC within the indicated concentration range of the sample DNA e Highly convenient assay with direct use of DNA isolated from cells or tissues no need for DNA digestion or hydrolysis e Universal positive and negative controls are included which are suitable for quantifying 5 fC from any species e Strip well microplate format makes the assay flexible manual or high throughput analysis e Simple reliable and consistent assay conditions PRINCIPLE amp PROCEDURE The MethylFlash 5 Formylcytosine 5 fC DNA Quantification Kit Colorimetric contains all the reagents necessary for the quantification of 5 fC In this assay DNA is bound to strip wells that are specifically treated to have a high DNA affinity 5 fC is detected using capture and detection antibodies The detected signal is enhanced and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer The amount of 5 fC is proportional to the OD intensity measured 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 Epigentek Group Inc All rights reserved Products are for research use only Printed 2015 08 20 P 1041 Prepare genomic DNA We recommend using Epigentek s series of DNA isolation kits Bind DNA to assay well
15. h Remove the Diluted ES solution from each well Wash each well with 150 ul of the Diluted WB each time for five times 4 Signal Detection a Add 100 ul of DS to each well and incubate at room temperature for 1 to 10 min away from light Begin monitoring color change in the sample wells and control wells The DS solution will turn blue in the presence of sufficient 5 fC Add 100 ul of SS to each well to stop enzyme reaction when color in the positive control wells turns medium blue Mix the solution by gently shaking the frame and wait 1 2 min to allow the color reaction to completely stop The color will change to yellow after adding SS and the absorbance should be read on a microplate reader at 450 nm within 2 to 15 min Note If the strip well plate frame does not fit in the microplate reader transfer the solution to a standard 96 well microplate 5 5 fC Calculation Relative Quantification To determine the relative 5 fC status of two different DNA samples simple calculation for the percentage of 5 fC in your total DNA can be carried out using the following formula Sample OD NC OD S 5fC x 10 PC OD NC OD P S is the amount of input sample DNA in ng P is the amount of input positive control PC in ng Example calculation Average OD450 of NC is 0 120 Average OD450 of PC is 0 320 Average OD450 of Sample is 0 141 S is 300 ng P is 0 2 ng 200 pg 0 141 0 120 300 5 fC
16. ire experiment experiment as different pipette devices may have slight variations in performance Capture Antibody vial Buffer evaporated due to the Add 1X PBS buffer into the Capture appears to be empty or very small volumes resulting in Antibody vial until you restore the correct insufficient in volume a higher concentrated antibody intended volume according to the Kit Contents described in this User Guide Mix and centrifuge prior to use RELATED PRODUCTS 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 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 Cell DNA Extraction Kit Methylated and Hydroxymethylated DNA Quantification P 1034 MethylFlash Methylated DNA Quantification Kit Colorimetric P 1035 MethylFlash Methylated DNA Quantification Kit Fluorometric P 1036 MethylFlash Hydroxymethylated DNA Quantification Kit Colorimetric P 1037 MethylFlash Hydroxymethylated DNA Quantification Kit Fluorometric Hydroxymethylated DNA Immunoprecip
17. istent timing in between each addition of solutions Color reaction is not evenly stopped due to an inconsistent order of adding solutions Ensure all solutions particularly DS Developer Solution and SS Stop Solution are added in the same order each time as all other solutions The solutions are not evenly added due to inconsistency in pipetting volume Ensure the solution in each pipette tip is equal in the multi channel pipette Equilibrate the pipette tip in any solutions before adding them Ensure the solutions especially those with small volumes e g 1 ul are completely added into the wells Solutions or antibodies were not actually added into the wells Do not allow pipette tip to touch the outer edges or inner sides of the wells to prevent solutions from sticking to the surface 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 11 Printed 2015 08 20 P 1041 Did not sufficiently shake the Gently and evenly shake the plate frame solutions in the wells after across a flat surface so that the solutions adding SS Stop Solution at Step in the wells are better distributed Do not 4b stir Did not use the same pipette Use the same multi channel pipette device throughout the device throughout the ent
18. itation P 1038 MethylFlash Hydroxymethylated DNA Immunoprecipitation hmeDIP Kit DNA Demethylation Enzyme Activity Assay P 3019 EpiQuik DNA Demethylase Activity Inhibition Assay Kit P 3086 Epigenase 5mC Hydroxylase TET Activity Inhibition Assay Kit Colorimetric P 3087 Epigenase 5mC Hydroxylase TET Activity Inhibition Assay Kit Fluorometric Page 12 Printed 2015 08 20 P 1041
19. l is not contaminated from accidentally adding sample or positive control DNA or from using contaminated tips Incubation time is too long The incubation time at Step 2d should not exceed 2 h Over development of color Decrease the development time in Step 4a before adding SS Stop Solution in Step 4b No signal or weak signal only in sample wells DNA sample is not properly extracted or purified Ensure the DNA sample is in good quality 260 280 ratio should be gt 1 7 with no or minimal RNA contamination Sample amount added into the wells is insufficient Ensure a sufficient amount of DNA is used as indicated in Step 2 Little or no 5 fC contained in the sample N A Uneven color development Insufficient washing of the wells Ensure the wells are washed according to the guidance of washing and residue washing buffer is removed as much as possible Delayed color development or delayed stopping of color development in the wells Ensure color development solution or stop solution is added sequentially and is consistent with the order in which you added the other reagents e g from well A to well G or from well 1 to well 12 Large variation between replicate wells Color reaction is not evenly stopped due to an inconsistency in pipetting time Ensure DS Developer Solution and SS Stop Solution are added at the same time between replicates or otherwise maintain a cons
20. p 2 The final amounts should be 10 20 40 100 200 and 400 pg per well 2 For optimal binding sample DNA volume added should not exceed 8 ul 3 To ensure that the NC Diluted PC and sample DNA are completely added into the wells the pipette tip should be placed in the BS Bind Solution in the well and aspirated in out 1 2 times d Cover strip plate with plate seal or Parafilm M and incubate at 37 C for 90 min e Remove the BS Binding Solution from each well Wash each well with 150 ul of the Diluted WB 1X Wash Buffer each time for three times This can be done by simply pipetting Diluted WB in and out of the wells 3 5 fC DNA Capture a Add 50 ul of the Diluted CA to each well then cover and incubate at room temperature for 60 min b Remove the Diluted CA solution from each well c Wash each well with 150 ul of the Diluted WB each time for three times d Add 50 ul of the Diluted DA to each well then cover and incubate at room temperature for 30 min e Remove the Diluted DA solution from each well f Wash each well with 150 ul of the Diluted WB each time for four times g Add 50 ul of the Diluted ES to each well then cover and incubate at room temperature for 30 min 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 08 20 Epigentek Group Inc All rights reserved Products are for research use only P 1041
Download Pdf Manuals
Related Search