User Manual
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1. EpiQuik Hydroxymethylated DNA Immunoprecipitation hMeDIP Kit Base Catalog P 1038 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiQuik Hydroxymethylated DNA Immunoprecipitation nMeDIP Kit is suitable for selective enrichment of DNA fragments containing 5 hydroxymethylcytosine in a high throughput format using DNA isolated from various species The hydroxymethylated DNA that is enriched with this kit can be used for various downstream applications including PCR nhMeDIP PCR and microarray AMeDIP chip Starting Material and Input Amount of DNA The starting material should be good quality purified DNA The amount of DNA for each reaction can be 0 1 ug approximately 1 x 10 cells to 1 ug For an optimal reaction the input DNA amount should be 0 5 ug per well DNA Shearing Genomic DNA should be sheared by sonication before starting hydroxymethylated DNA immunoprecipitation The sheared DNA fragments should range in size from 200 600 base pairs For the best results and your convenience we recommend using the EpiSonic Multi Functional Bioprocessor 1100 which allows for simultaneous sonication of multiple samples 1 384 in sealed vials for consistent and desired size range of DNA 200 600 base pairs Internal Control Negative Non Immune IgG and positive controls Control DNA are provided in this kit The Control DNA is a 200 base pair DNA fragment containing 44 cytosine residues which are hydroxymethylated The2. Incorrect temperature and or insufficient time during DNA release Ensure the incubation time and temperature described in the protocol are followed correctly Improper PCR program settings Ensure PCR program settings are properly programmed Inappropriate PCR reaction solution If using a homemade PCR reaction solution check if each component is correctly mixed If using a PCR Fast Kit check if it is suitable for your PCR Inappropriate primers Confirm the species specificity of your primers Primers should be designed to cover a short sequence region 70 150 bp for more efficient and exact amplification of target DNA regions Improper sample storage DNA samples should be stored at 20 C 3 6 months No difference in signal intensity between negative control and positive control Insufficient washing of wells Check if washing recommendations at each step is performed according to the protocol If the signal intensity in the negative control is still high washing stringency can be increased in the following ways 1 Increase wash time at each wash step after adding Diluted WB leave it in the tubes wells for 2 3 min before removing it 2 Add an additional one or two wash steps The volume of Diluted WB is sufficient for at least two extra washes for each sample Too many PCR cycles Pleateu phase of amplification caused by over increased number of PCR cycles i
3. primer solution and DNA template Mix well by vortexing briefly Keep components on ice while in use Wand return to 20 C immediately following use Add components into each well according to the following Component Size ul 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 HzO 6 7 ul Total Volume 20 ul For the negative control use DNA RNA free water instead of DNA template Program the PCR Reactions Place the reaction plate in the instrument and set the PCR conditions as follow Cycle Step Temp Time Cycle Activation 95 C 7 min 1 95 C 10 sec Cycling 55 C 10 sec 40 72 C 8 sec Final Extension 72 1 min 1 110 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 9 Printed 2014 09 22 P 1038 Fold Enrichment Calculation Fold enrichment FE can be calculated by simply using a ratio of amplification efficiency of the ChIP sample over that of non immune IgG Amplification efficiency of Polymerase RNA II can be used as a positive control FE 28G CT Sample CT y 100 For example if CT for IgG is 38 and the sample is 34 then FE 208 39 x 100 1
4. 0 5 ml vial label as input DNA and place on ice Cover the wells with parafilm M and incubate at room temperature for 90 min on an orbital shaker at 50 100 rpm 5 Wash of the Reaction Wells a b Cc Carefully remove and discard the solution containing the reagents by pipetting out each well Thoroughly wash each well five times with 200 ul of the Diluted WB each time This can be done by simply pipetting Diluted WB in and out of the wells Wash each well with 200 ul of DRB one time by pipetting DRB in and out 6 Release and Elution of DNA a Prepare DRB PK Solution by adding 1 ul of Proteinase K to every 39 ul of DRB Mix b Add 40 ul of the DRB PK Solution to each well c Separate and insert the wells into a thermalcycler with a 48 well block d Tightly seal the wells with Adhesive 8 Well Strip Film and incubate at 60 C for 15 min followed by incubation at 95 C for 3 min Note If only a thermalcycler with a 96 well block is available then 1 incubate the wells at 65 C for 20 min and quickly transfer the DNA solution from each well to 0 2 ml strip PCR tubes Cap the PCR tubes and then incubate the PCR tubes containing the DNA solution at 95 for 3 min in the thermalcycler then 2 place the PCR tubes in room temperature If liquid is collected on the inside of the caps briefly centrifuge the liquid down to the bottom DNA is now ready for use or storage at 20 C For real time PCR analysis we recomme
5. WB 10X Wash Buffer to 180 ml of distilled water pH 7 2 7 5 This Diluted WB 1X Wash Buffer can now be stored at 4 C for up to six months 2 Preparation of Antibody Coated Wells 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 100 ul of AB to each well and then add the following antibodies 1 ul of Non Immune IgG to the negative control well 1 ul of 5 hmC Antibody to the sample wells and 1 ul of 5 hmC Antibody to the positive control wells Cover the wells with parafilm M and incubate at room temperature for 60 min Meanwhile prepare fragmented DNA as described in the next step 3 Shearing of Genomic DNA For the best results DNA should be fragmented by a suitable sonication method Probe based Sonication You will need to optimize the sonication settings For example DNA of 200 1000 bp size can be obtained by sonicating 3 4 pulses of 10 12 sec each at level 2 using a Branson Microtip probe followed by a 30 40 sec rest period on ice between each pulse Waterbath based Sonication Follow the pre optimized protocol in the EpiSonic Multi Functional Bioprocessor 1100 user manual for DNA shearing at a size range of 100 600 bp Note If desired remove 10 ul of sheared DNA for purification and agarose gel analysis along with a DNA marker on a 1 2 agarose gel stained wit
6. kit also includes control PCR primers that can be used for verifying the enrichment efficiency of hydroxymethylated control DNA Antibody The 5 hydroxymethylcytosine rabbit polyclonal antibody used in this kit is highly specific against hydroxymethylated DNA fragments both single and double stranded and is not cross reactive to methylated and unmethylated DNA fragments 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 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 1038 KIT CONTENTS Component 24 Reactions 48 Reactions 96 Reactions Storage Cat P 1038 24 Cat P 1038 48 Cat P 1038 96 Upon Receipt WB 10X Wash Buffer 5 ml 10 ml 20 ml 4 C AB Antibody Buffer 4ml 8 ml 16 ml RT HS hMeDIP Solution 3 ml 6 ml 12 ml RT DRB DNA Release Buffer 7 mi 14 ml 28 ml RT Non Immune IgG 0 6 mg ml 10 ul 20 ul 40 ul 47 5 hmC Antibody 0 6 mg ml 25 ul 50 ul 100 ul 4 C Control DNA 500 ng ml 5 ul 10 ul 20 ul 20 C Pro
7. 600 Endpoint PCR Primer Design Primers designed should meet the criteria for endpoint PCR For example the covered sequence region should be 100 400 bp in length PCR primer design tools e g Primer3Plus can be used to help in the selection of appropriate primer pairs PCR Reaction Endpoint PCR can be performed using your own proven method It is important to stop the PCR reaction at the exponential phase by setting up an appropriate number of PCR cycles in order to make a reliable comparision of enrichment efficiency obtained from different ChIP reactions Thus the optimized number of PCR cycles should be determined empirically PCR Product Analysis Endpoint PCR products can be analyzed by separating amplicons on a 1 2 agarose gel followed by staining with ethidium bromide and visulizing with UV illumination RELATED PRODUCTS Methylated DNA Immunoprecipitation P 1015 Methylamp Methylated DNA Capture Kit P 2019 EpiQuik Methylated DNA Immunoprecipitation Kit P 2020 EpiQuik Tissue Methylated DNA Immunoprecipitation Kit DNA Hydroxymethylation Analysis P 1036 MethylFlash Hydroxymethylated DNA Quantification Kit Colorimetric P 1037 MethylFlash Hydroxymethylated DNA Quantification Kit Fluorometric Sonication Instruments EQC 1100 EpiSonic Multi Functional Bioprocessor 1100 PCR Analysis P 1029 EpiQuik Quantitative PCR Fast Kit 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 10 Tel 1 87
8. 7 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 1038
9. articularly in brain specific gene regulation For example it was shown that 5 hmC inhibits the binding of the methyl CpG binding domain proteins to DNA suggesting a potential gene regulatory function of 5 hmC 5 hmC was observed to be linked with epigenetic reprogramming in mammalian zygotes However the exact functions of 5 hmC have not yet been fully identified since gene specific distribution of 5 hmC is unknown due to the inability of currently used DNA methylation analysis methods in distinguishing 5 hmC from 5 mC Because of the presence of 5 hmC in DNA with unclear functions in gene regulation and because of the discovery of enzymes that produce 5 hmC it is crucial to identify hydroxymethylation status in specific gene loci which would help to better understand methylation based epigenetic regulation of gene functions To achieve this Epigentek has developed an innovative method to capture DNA fragments containing 5 hmC and incorporated this method into its EpiQuik Hydroxymethylated DNA Immunoprecipitation nMeDIP Kit This kit uses a high affinity 5 hmC antibody to selectively capture double stranded or single stranded DNA fragments containing 5 hmC The kit has the following features e Extremely fast and convenient protocol with a total procedure time from input sample to ready to use hydroxymethylated DNA of less than 3 hours which includes a minimal handling time of less than 20 minutes e Flexible 96 stripwell microplate f
10. e for research use only Page 2 Printed 2014 09 22 P 1038 O Aerosol resistant pipette tips O Parafilm M O 0 2mlor 0 5 ml PCR vials GENERAL PRODUCT INFORMATION Quality Control Each lot of the EpiQuik Hydroxymethylated DNA Immunoprecipitation hMeDIP Kit 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 EpiQuik Hydroxymethylated DNA Immunoprecipitation hMeDIP Kit is for research use only and is not intended for diagnostic or therapeutic applications Intellectual Property The EpiQuik Hydroxymethylated DNA Immunoprecipitation hMeDIP Kit and the method of processing contain proprietary technologies by Ep
11. entek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 1038 be used for various downstream applications including PCR nhMeDIP PCR and microarray nMeDIP chip DNA shearing hmeDNA antibody immunoprecipitation Release of hmeDNA from hmeDNA Ab complex Elution of hmeDNA PCR microarray or similar downstream application Schematic procedure of the EpiQuik Hydroxymethylated DNA Immunoprecipitation hMeDIP Kit 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 1200 1000 800 5 600 Fold Enrichment 400 200 0 T T T Non immune IgG Unmethylated Methylated DNA hmeDNA DNA Fig 1 Selective enrichment of hydroxymethyated DNA with the EpiQuik hMeDIP Kit 50 pg of unmethylated methylated and hydroxymethylated DNA control were each spiked into fragmented human genomic DNA 500 ng hMeDIP was processed with the 5 hmC antibody and non immune IgG included in the kit Eluted DNA was analyzed by real time PCR with the control primers included in the kit to detect the presence of spiked control DNA Fold enrichment represents the amount of recovered control DNA and was calculated based on the Cts 120 GAPDH 100 BOcT4 re c E 80 E 2 pm w so 60 D 2
12. h ethidium bromide Visualize it under ultraviolet light 4 Preparation of hMeDIP Reaction a Remove AB from the wells and wash the wells two times with 200 ul of Diluted WB each time 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 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 1038 b Dilute the Control DNA to 50 ng ml 50 pg ul by adding 1 ul of Control DNA to 9 ul of HS hMeDIP Solution and dilute your sample DNA with HS hMeDIP Solution to 10 ug ml 10 ng ul Setup the hMeDIP reactions by adding the appropriate reagents to each corresponding well according to the following chart Reagent Sample Well Positive Negative Control Negative Control Well Control Well Well For Sample For Control DNA HS Solution 50 ul 99 ul 50 ul 99 ul Sample DNA 50 ul N A 50 ul N A Control DNA Oul 1 ul Oul 1 ul Note 1 The final amount of each component should be 500 ng well for sample DNA and 50 pg well for contro DNA 2 An input DNA control is only used for estimating enrichment efficiency of hMeDIP and is generally not needed as the included positive and negative controls can be used for estimating the same objective more accurately 3 If an input DNA control is to also be included remove 5 ul of the sonicated DNA solution prepared at Step 3 to a
13. igentek 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 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 amajor epigenetic modification in phenotype and gene expression has been widely recognized Quite recently a novel modified nucleotide called 5 hydroxymethyl cytosine 5 hmC has been detected to be abundant in mouse brains and embryonic stem cells In mammals it can be generated by the 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 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 1038 Ht HH Hae H oy 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 A line of evidence showed that 5 hmC also plays an important and different role from 5 mC in regulation of DNA methylation chromatin remodeling and gene expression p
14. n endpoint PCR may mask the difference in 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 8 Printed 2014 09 22 P 1038 signal intensity between negative contol and positive control Decreasing the number of PCR cycles ex 32 35 cycles to keep ampification at exponential phase will reduce high background in endpoint PCR and allow differences in amplification to be seen Real time PCR is another alternative in such cases Little or No PCR products PCR conditions are not Make sure the PCR conditions include generated from positive optimized correct and appropriate temperature cycles control only and solutions PCR ANALYSIS Real Time PCR Primer Design Primers designed should meet the criteria for real time PCR For example the covered sequence region should be 50 150 bp in length G C stretches at 3 ends of primers should be avoided PCR Reaction Real time PCR can be performed using your own proven method For your convenience and best results Epigentek offers the EpiQuik Quantitative PCR Fast Kit Cat P 1029 which is optimized for fast GPCR reactions As an example the protocol is presented below Prepare the PCR Reactions Thaw all reaction components including master mix DNA RNA free water
15. nd using 1 2 ul of eluted DNA in a 20 ul PCR reaction Input DNA can be added directly to a PCR reaction after appropriate dilution For end point PCR the number of PCR cycles may need to be optimized for better PCR results In general the amplification difference between non immune IgG control and 5 hmC antibody may vary from 3 to 8 cycles depending on experimental conditions 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 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 1038 For hMeDIP chip additional DNA cleanup concentration and whole genome amplification WGA steps may be needed For your convenience Epigentek offers the DNA Concentrator Kit Cat No P 1006 for DNA cleanup and concentration TROUBLESHOOTING Problem Possible Cause Suggestion Little or no PCR products generated from samples Poor DNA quality due to insufficient cell amounts extraction or degradation To obtain the best results the amount of DNA per hMeDIP should be 0 1 1 ug with 260 280 ratio gt 1 6 Inappropriate DNA fragmenting conditions DNA fragment size should be between 200 1000 bp with an optimal size range of 200 600 bp Oversized DNA fragments may reduce targeted DNA capturing via antibody and undersized DNA fragments may decrease PCR efficiency
16. ormat makes the assay very easy to handle manual method with one reaction at a time or high throughput method with 96 reactions at a time Highly efficient enrichment ratio of positive negative control gt 1000 Low DNA input requirement of as low as 0 1 ug per reaction High reproducibility through pre optimized hMeDIP conditions Compatible with various downstream analysis workflows including hMeDIP PCR and hMeDIP chip PRINCIPLE amp PROCEDURE The EpiQuik Hydroxymethylated DNA Immunoprecipitation hMeDIP Kit contains all reagents required for carrying out a successful hMeDIP procedure using DNA isolated from mammalian cells or tissues This kit includes a positive control DNA fragment a negative control non immune IgG and control primers that can be used with the positive control to demonstrate the enrichment efficacy for hydroxymethylated DNA with the kit reagents and protocol The positive control DNA containing 5 hmC can be immunoprecipitated by a 5 hmC antibody but not by a non immune IgG In this hMeDIP immunoprecipitation of 5 hmC enriched DNA fragments is processed in a microplate under optimized reaction conditions which enables hMeDIP to be completed within 3 hours with high efficiency Immunoprecipitated hydroxymethylated DNA is then cleaned released and eluted Eluted DNA can 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 epig
17. teinase K 10 mg ml 28 ul 56 ul 112 ul 4 C Control Primer Forward 20 uM 5 ul 10 ul 20 ul 4 C Control Primer Reverse 20 uM 5 ul 10 ul 20 ul 4 C 8 Well Assay Strips With Frame 3 6 12 4 C Adhesive 8 Well Strip Film 3 6 12 RT User Guide 1 1 1 RT Spin the solution down to the bottom prior to use 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 Control DNA at 20 C away from light 2 Store WB Non Immune IgG 5 hmC Antibody Proteinase K Control Primer Forward Control Primer Reverse and 8 Well Assay Strips at 4 C away from light 2 Store remaining components at room temperature away from light All components of the kit are stable for 6 months from the date of shipment when stored properly Note Check if WB 10X Wash Buffer contains salt precipitates before use If so briefly warm at room temperature or 37 C and shake the buffer until salts are re dissolved MATERIALS REQUIRED BUT NOT SUPPLIED O Variable temperature waterbath or incubator oven Thermalcycler with 48 or 96 well block Sonication device Orbital shaker O oO eo Adjustable pipette and multiple channel pipette 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 ar
18. w 40 e 20 0 ad Non immune IgG 5 hmC Ab Fig 2 Sensitive detection of gene specific hydroxymethylation by hMeDIP QPCR Human brain DNA 500 ng was fragmented to 200 600 bps with an EpiSonic 1000 The fragmented DNA was used for hydroxymethylated DNA enrichment with the EpiQuik hMeDIP Kit Eluted DNA was analyzed by real time PCR with primers specifically for OCT4 or GAPDH sequences in the promoter regions Results show that the promoter region is hydroxymethylated in OCT4 but not in GAPDH Fold enrichment represents the amount of recovered DNA and was calculated based on the Cts Page 5 Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 1038 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 1 ug per reaction An optimal amount is 500 ng per reaction 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 short term or 20 C long term until use 1 Preparation of 1X Wash Buffer 24 Reaction Kit Add 5 ml of WB 10X Wash Buffer to 45 ml of distilled water pH 7 2 7 5 48 Reaction Kit Add 10 ml of WB 10X Wash Buffer to 90 ml of distilled water pH 7 2 7 5 96 Reaction Kit Add 20 ml of
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