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MethylFlash™ Methylated DNA Quantification Kit
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1. Methylated DNA Bind DNA to assay well Unmethylated DNA Wash wells then add capture antibody OD450 nm Wash wells then add detection antibody and enhancer solution 0 0205 1 2 5 10 100 Add color developing solution for color develop Input DNA ng ment then measure absorbance Demonstration of high sensitivity and specificity of methylated DNA detection achieved by the MethylFlash kit Synthetic Schematic procedure of the MethylFlash unmethylated DNA contains 50 of cytosine and methylated DNA Methylated DNA Quantification Kit contains 50 of 5 methylcytosine were added into the assay wells Colorimetric at different concentrations and then measured with the MethviFlash Methviated DNA Quantification Kit Colorimetric ASSAY PROTOCOL Starting Materials Inout DNA Amount DNA amount can range from 50 ng to 200 ng per reaction An optimal 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 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 2014 06 19 Epigentek Group Inc All rights reserved Products are for research use only P 1034 DNA Storage lsolated genomic DNA can be stored a
2. MethylFlash Methylated DNA Quantification Kit Colorimetric Base Catalog P 1034 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The MethylFlash Methylated DNA Quantification Kit Colorimetric is suitable for detecting global DNA methylation status using DNA isolated from any species such as mammalians plants fungi bacteria and viruses in a variety of forms including but not limited to cultured cells fresh and frozen tissues paraffin embedded tissues plasma serum samples and body fluid samples Input DNA The amount of DNA for each assay can be 50 to 200 ng For optimal quantification the input DNA amount should be 100 ng as methylated DNA varies from tissue to tissue and can be less than 1 of total DNA in some species Starting Materials Starting materials can include various tissue or cell samples such as cells from flask or microplate cultured cells fresh and frozen tissues paraffin embedded tissues plasma serum samples body fluid samples etc Internal Control Both negative and positive DNA controls are provided in this kit A standard curve can be performed range 1 to 10 ng or a single quantity of methylated DNA can be used as a positive control Because global methylation can vary from tissue to tissue and from normal and diseased states it is advised to run replicate samples to ensure that the signal generated is validated This kit will allow the user to quantify an absolute amount of methylated DNA an
3. P 1017 P 1018 FitAmp General Tissue Section 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 FitAmp Blood and Cultured Cell DNA Extraction Kit DNA Methylation Quantification P 1035 P 1036 P 1037 MethylFlash Methylated DNA Quantification Kit Fluorometric MethylFlash Hydroxymethylated DNA Quantification Kit Colorimetric MethylFlash Hydroxymethylated DNA Quantification Kit Fluorometric 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 11 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 06 19 Epigentek Group Inc All rights reserved Products are for research use only P 1034
4. Sample4 Sample8 Sample 12_ Sample 16 Sample 20 E Sample2 Sample5 Sample9 Sample 13 Sample 17 Sample 21 F Sample2 Sample5 Sample9 Sample 13 Sample 17 Sample 21 G Sampe3 Sample6 Sample 10 Sample 14 Sample 18 Sample 22_ H Sampe3 Sample6 Sample 10_ Sample 14 Sample 18 Sample 22 Table 2 Standard Curve Preparation The suggested strip well plate setup for standard curve preparation in a 48 assay format for a 96 assay format Strips 7 to 12 can be configured as Sample The controls and samples can be measured in duplicate Weli Strip1 Strip2 Strip3 Strip4 Strip5 Strip6 A MES ME3 Sampe3 Sample7 Sample11 Sample 15 B ME405ng ME40 5ng Sample3 Sample7 Sample 11 Sample 15 _ D ME42 0ng ME420ng Sample4 Sample8 Sample 12 Sample 16 _ E ME450ng ME450ng Sampe5 Sample9 Sample 13 Sample 17 F ME410 0ng ME410 0ng Sample5 Sample9 Sample 13 Sample 17 _ G Sample Sampe2 Sampe6 _ Sample10 Sample 14 Sample 18 _ H Sample 1 Sample 2 Sample 6 Sample 10 Sample 14 Sample 18 TROUBLESHOOTING Problem No signal in both the positive control and sample wells Reagents are added incorrectly The well is incorrectly washed before DNA binding The bottom of the well is not completely covered by the ME2 Binding Solution Incubation time and temperature are incorrect 110 Bi County Blvd Ste 122 Farmingdale N
5. several times Ensure the solution coats the bottom of the well evenly Note 1 For a single point control add 1 ul of ME4 at a concentration of 5 ng ul as prepared in Step 2 for the standard curve add 1 ul of Diluted ME4 at concentrations of 0 5 to 10 ng ul see the chart in Step 2 The final amounts should be 0 5 1 2 5 and 10 ng per well 2 For optimal binding sample DNA volume added should not exceed 8 ul 3 To ensure that ME3 Diluted ME4 and sample DNA are completely added into the wells the pipette tip should be placed into the ME2 solution in the well and aspirated in out 1 2 times 4 Add replicate samples to the same column according to the suggested strip well set up in Table 1 or Table 2 Cover strip plate with plate seal or Parafilm M and incubate at 37 C for 90 min 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 Printed 2014 06 19 Epigentek Group Inc All rights reserved Products are for research use only P 1034 e Remove the binding reaction solution from each well Wash each well three times with 150 ul of Diluted ME1 1X Wash Buffer To wash the wells pipette Diluted ME1 1X Wash Buffer into the wells then remove it from the wells and discard it Repeat for a total of three washes 4 Methylated DNA Capture a Dilute ME5 at 1 1000 dilution with Diluted ME1 b Add 50 ul of the Diluted ME5 t
6. Y 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 Possible Cause Suggestion Check if reagents are added in the proper order and if any steps in the protocol may have been omitted by mistake Ensure the well is not washed prior to adding the positive control and sample Ensure the solution coats the bottom of the well by gently tilting from side to side or shaking the plate several times Ensure the incubation time and temperature described in the protocol are followed correctly Page 9 Printed 2014 06 19 P 1034 Insufficient input materials Ensure that a sufficient amount of positive control gt 1 ng and samples Incorrect absorbance reading Kit was not stored or handled properly No signal or weak signal in only the positive control wells The ME4 Positive Control is degraded due to improper storage conditions High background present in the negative control wells Insufficient washing of wells Contaminated by sample or positive control DNA Incubation time is too long Over development of color Large variation between replicate wells Color reaction is not evenly stopped due to an inconsistency in pipetting time Color reaction is not evenly stopped due to an inconsistent order of adding solutions The solutions are not evenly added du
7. d determine the relative methylation 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 2014 06 19 Epigentek Group Inc All rights reserved Products are for research use only P 1034 Kit Contents Cat P 1034 48 Cat P 1034 96 Upon Receipt Ewet Assay Sips With Fame 6 e qo Spin the solution down to the bottom prior to use Note The ME3 Negative Control is an unmethylated polynucleotide containing 50 of cytosine The ME4 Positive Control is a methylated polynucleotide containing 50 of 5 methylcytosine 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 ME3 ME4 ME6 and ME7 at 20 C away from light 2 Store ME1 ME5 ME8 and 8 Well Assay Strips at 4 C away from light 8 Store remaining components ME2 and MEQ at room temperature away from light All components of the kit are stable for 6 months from the date of shipment when s
8. d 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 methylated DNA 5 mC in total DNA using the following formulas Sample OD ME3 OD 5 mC ng Slope x 2 5 mC Amount ng 5 nmC X100 S S is the amount of input sample DNA in ng 2 is a factor to normalize 5 mC in the positive control to 100 as the positive control contains only 50 of 5 mC Example calculation Average OD450 of ME3 is 0 075 Average OD450 of sample is 0 475 Slope is 0 12 OD ng S is 100 ng 0 475 0 075 5 mC ng E 1 67 ng 0 12 X 2 1 67 5 mC m xX 100 1 67 100 110 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 Printed 2014 06 19 Epigentek Group Inc All rights reserved Products are for research use only P 1034 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 for a 96 assay format Strips 7 to 12 can be configured as Sample The controls and samples can be measured in duplicate Well Strip1 Strip2 Strip3 Strip4 Strip5 Strip6 A MES ME3 Sample7 Sample 11_ Sample 15_ Sample 19_ B _ ME4 ME4__ Sample7 Sample 11_ Sample 15_ Sample 19_ D Sampe1
9. e to inconsistency in pipetting volume Solutions or antibodies were not actually added into the wells 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 gt 100 ng is added into the wells Check if appropriate absorbance wavelength 450 nm is used Ensure all components of the kit were stored at the appropriate temperature and the cap is tightly capped after each Opening or use The positive control DNA is insufficiently Ensure a sufficient amount of positive added to the well in Step 3c control DNA is added Follow the Shipping amp Storage guidance in this User Guide for storage of ME4 Positive Control Check if washing recommendations at each step is performed according to the protocol Ensure the well is not contaminated from adding sample or positive control DNA accidentally or from using contaminated tips The incubation time at Step 3d should not exceed 2 h Decrease the development time in Step 5a before adding ME9 Stop Solution in Step 5b Ensure ME8 Developer Solution and ME9 Stop Solution is added at the same time between replicates or otherwise maintain a consistent timing in between each addition of solutions Follow the suggested strip well setup to reduce variability between replicates Ensure all solu
10. ell demonstrated that the decrease in global DNA methylation is one of the most important characteristics of cancer Thus the quantification of 5 mC content or global methylation in cancer cells could provide very useful information for detection and analysis of this disease Quite recently a novel modified nucleotide 5 hydroxymethylcytosine 5 hmC has been detected to be abundant in mouse brain and embryonic stem cells In mammals it can be generated by oxidation of 5 methylcytosine a reaction mediated by the Tet family of enzymes and Dnmt proteins It is a hydroxylated and methylated form of cytosine 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 2014 06 19 Epigentek Group Inc All rights reserved Products are for research use only P 1034 Unmethylated DNA Methylated DNA Hydroxymethylated DNA T C G T C G A C G T C G T C G A C G T C G T C G A C G The broader functions of 5 hmC in epigenetics are still a mystery today However a line of evidence does show that 5 hmC plays a role in DNA methylation structures and patterns Because of the presence of both 5 mC and 5 hmC in DNA with possibly different functions it is important to determine the contents of these two modified nucleotides and their ratios in different cell types and in different compartments of the genome of mammalians It is particularly impor
11. o each well then cover and incubate at room temperature for 60 min c Remove the Diluted ME5 solution from each well d Wash each well three times with 150 ul of Diluted ME1 1X Wash Buffer e Dilute ME6 at 1 2000 dilution with the Diluted ME1 h Add 50 ul of the Diluted ME6 to each well then cover and incubate at room temperature for 30 min g Remove the Diluted ME6 solution from each well Wash each well four times with 150 ul of Diluted ME1 1X Wash Buffer Dilute ME7 at 1 5000 dilution with the Diluted ME1 Add 50 ul of the Diluted ME7 to each well then cover and incubate at room temperature for 30 min Aes k Remove the Diluted ME7 solution from each well Wash each well five times with 150 yl of Diluted ME1 1X Wash Buffer 5 Signal Detection a Add 100 ul of ME8 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 ME8 solution will turn blue in the presence of sufficient methylated DNA b Add 100 ul of ME9 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 be completely stopped The color will change to yellow after adding ME9 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
12. ontrols are included which are suitable for quantifying methylated DNA from any species e Strip well microplate format makes the assay flexible manual or high throughput analysis References Robertson K D Nat Rev Genet 6 597 610 2005 Kriaucionis S et al Science 324 929 930 2009 Wyatt G R et al Biochem J 55 774 8 1953 Tahiliani M et al Science 324 930 935 2009 Valiniuck V et al Nucleic Acids Res 32 4100 4108 2004 Valinluck V et al Cancer Res 67 946 50 2007 Jin S G et al Nucleic Acids Res 38 e125 2010 aoe ON gt 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 06 19 Epigentek Group Inc All rights reserved Products are for research use only P 1034 PRINCIPLE amp PROCEDURE The MethylFlash Methylated DNA Quantification Kit Colorimetric contains all reagents necessary for the quantification of global DNA methylation In this assay DNA is bound to strip wells that are specifically treated to have a high DNA affinity The methylated fraction of DNA is detected using capture and detection antibodies and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer The amount of methylated DNA is proportional to the OD intensity measured Prepare genomic DNA We recommend using Epigentek s series of DNA isolation kits a
13. 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 Methylated DNA Quantification Kit Colorimetric is for research use only and is not intended for diagnostic or therapeutic applications Intellectual Property The MethylFlash Methylated 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 5 mC In somatic cells 5 mC is found almost exclusively in the context of paired symmetrical methylation of the dinucleotide CpG whereas in embryonic stem ES cells a substantial amount of 5 mC is also observed in non CpG contexts The biological importance of 5 mC as a major epigenetic modification in phenotype and gene expression has been recognized widely For example global decrease in 5 mC content DNA hypomethylation is likely caused by methyl deficiency due to a variety of environmental influences and has been proposed as a molecular marker in multiple biological processes such as cancer It has been w
14. t 4 C short term or 20 C long term until use 1 Preparation of 1X Wash Buffer ME1 48 Assays Kit Add 13 ml of ME1 10X Wash Buffer to 117 ml of distilled water pH 7 2 7 5 96 Assays Kit Add 26 ml of ME1 10X Wash Buffer to 234 ml of distilled water pH 7 2 7 5 Note This Diluted ME1 1X Wash Buffer can now be stored at 4 C for up to six months All other diluted solutions should be kept on ice at all times and should be discarded if not used within the same day 2 Preparation of Diluted Positive Control ME4 single Point Control Preparation Dilute ME4 Positive Control with 1X TE to 5 ng ul 1 ul of ME4 3 ul of TE Suggested Standard Curve Preparation First dilute ME4 to 10 ng ul 5 ul of ME4 5 ul of 1X TE Then further prepare five different concentrations with the 10 ng ul diluted ME4 and 1X TE into 0 5 1 2 5 and 10 ng ul according to the following dilution chart Resulting ME4 Tube ME4 10ng pl 1XTE Concentration 3 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 Add 80 ul of ME2 Binding Solution to each well Add 1 ul of MES 1 ul of Diluted ME4 see note below and 100 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
15. tant to identify that in healthy and diseased human cell tissues the epigenetic change at the DNA level is due to methylation or hydroxymethylation Several chromatography based techniques such as HPLC TLC mass spectrometry are used for detecting 5 mC and 5 hmC However these methods are time consuming and have low throughput with high costs To address this problem Epigentek offers the MethylFlash Methylated DNA Quantification Kit Colorimetric to quantify 5 mC or methylated DNA This kit is optimized for paired use with our Methy Flash Hydroxymethylated DNA Quantification Kit for simultaneously quantifying both methylated and hydroxymethylated DNA or for quantifying methylated DNA by itself 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 finished within 4 hours e Innovative kit composition enables background signals to be extremely low which eliminates the need for plate blocking and allows the assay to be simple accurate reliable and consistent e High sensitivity of which the detection limit can be as low as 0 2 ng of methylated DNA methylation e Optimized antibody and enhancer solutions allow high specificity to 5 mC with no cross reactivity to unmethylated cytosine and no or negligible cross reactivity to hydroxymethylcytosine within the indicated concentration range of the sample DNA e Universal positive and negative c
16. the microplate reader transfer the solution to a standard 96 well microplate 6 5 mC Calculation Relative Quantification To determine the relative methylation status of two different DNA samples simple calculation of percentage of 5 mC in total DNA can be carried out using the following formula Sample OD ME3 OD S 5 mC eo X 100 ME4 OD ME3 OD x 2 P S is the amount of input sample DNA in ng 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 2014 06 19 Epigentek Group Inc All rights reserved Products are for research use only P 1034 P is the amount of input positive control ME4 in ng 2 Is a factor to normalize 5 mC in the positive control to 100 as the positive control contains only 50 of 5 mC Example calculation Average OD450 of MES is 0 075 Average OD450 of ME4 is 0 675 Average OD450 of Sample is 0 475 S is 100 ng Pis 5ng 0 475 0 075 100 5 mC x 1003 1 673 0 6750 0 075 X2 3 Absolute Quantification To quantify the absolute amount of methylated DNA using an accurate calculation first generate a standard curve and plot the OD values versus the amount of ME4 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 an
17. tions particularly ME8 Developer Solution and MEY Stop Solution are added in the same order each time as all other solutions 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 Do not allow pipette tip to touch the outer edges or inner sides of the wells to prevent solutions from sticking to the Page 10 Printed 2014 06 19 P 1034 Capture antibody vial appears to be empty or insufficient in volume Did not sufficiently shake the solutions in the wells evenly after adding ME9 Stop Solution in Step 5b Did not use the same pipette device throughout the experiment Buffer evaporated due to the very small volumes resulting in a higher concentrated antibody surface Gently and evenly shake the plate frame across a flat surface so that the solutions in the wells are better distributed Do not Stir Use the same multi channel pipette device throughout the entire experiment as different pipette devices may have slight variations in performance Add 1X PBS buffer into the Capture Antibody vial until you restore the correct intended volume according to the Kit Contents described in this User Guide Mix and centrifuge prior to use RELATED PRODUCTS DNA Sample Preparation P 1003 P 1004 P 1006 P 1007 P 1009
18. tored properly Note Check ME1 10X Wash Buffer contains salt precipitates before use If so briefly warm at room temperature or 37 C and shake the buffer until the salts are re dissolved MATERIALS REQUIRED BUT NOT SUPPLIED O Adjustable pipette multi channel recommended Aerosol resistant pipette tips Microplate reader capable of reading absorbance at 450 nm 1 5 ml microcentrifuge tubes Oo O 8 Incubator for 37 C incubation 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 2014 06 19 Epigentek Group Inc All rights reserved Products are for research use only P 1034 Plate seal or Parafilm M Distilled water 1X TE buffer pH 7 5 to 8 0 0 OF 0 o0 Isolated DNA of interest GENERAL PRODUCT INFORMATION Quality Control Each lot of the MethylFlash Methylated DNA Quantification Kit 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
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