hMeDIP - Abcam
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1. 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 C for 3 min in the thermalcycler 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 recommend 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 EMH5 and EMH6 may vary from 3 to 8 cycles depending on experimental conditions For hMeDIP chip additional DNA cleanup concentration and whole genome amplification WGA steps may be needed 17 5 Troubleshooting Problem Cause Solution 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 rang2. 0 uL N A EMH7 O uL 1 uL O uL 1 uL Note 1 The final amount of each component should be 500 ng well for sample DNA and 50 pg well for control 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 D to a 0 5 ml vial label as input DNA and place on ice 3 Cover the wells with parafilm M and incubate at room temperature for 90 min on an orbital shaker at 50 100 rpm 15 F Wash of the Reaction Wells 1 Carefully remove and discard the solution containing the reagents by pipetting out each well 2 Thoroughly wash each well five times with 200 ul of the Diluted EMH1 each time This can be done by simply pipetting Diluted EMH1 in and out of the wells 3 Wash each well with 200 ul of EMH4 one time by pipetting EMH4 in and out G Release and Elution of DNA 1 Prepare EMH4 EMH8 Solution by adding 1 ul of EMH8 to every 39 ul of EMH4 Mix 2 Add 40 ul of the EMH4 EMH8 Solution to each well 3 Separate and insert the wells into a thermalcycler with a 48 well block 4 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 16 Note
3. A Immunoprecipitation MeDIP Kit Tissue ab117136 EpiSeeker hydroxymethylated DNA Quantification Kit Colorimetric ab1 17130 EpiSeeker hydroxymethylated DNA Quantification Kit Fluorometric ao117131 21 22 abcam discover more UK EU and ROW Email technical abcam com Tel 44 0 1223 696000 www abcam com US Canada and Latin America Email us technical abcam com Tel 888 77 ABCAM 22226 www abcam com China and Asia Pacific Email hk technical abcam com Tel 108008523689 Edi www abcam cn Japan Email technical abcam co jp Tel 81 0 3 6231 0940 www abcam co jp Copyright 2012 Abcam All Rights Reserved The Abcam logo is a registered trademark All information detail is correct at time of going to print
4. P chip 1200 1000 800 600 Fold Enrichment 400 200 0 NonimmuneigG Unmethylated Methylated DNA hmeDNA DNA Selective enrichment of hydroxymethyated DNA with ab117134 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 4 BOCT4 Relative Fold Enrichment a S Non immune IgG 5 hmC Ab Sensitive detection of gene specific hydroxymethylation by hMeDIP QPCR Human brain DNA 500 ng was fragmented to 200 600 bps with a sonicator The fragmented DNA was used for hydroxymethylated DNA enrichment with the 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 ab117134 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 enrich
5. abcam discover more ab117134 EpiSeeker Hydroxymethylated DNA Immunoprecipitation hMeDIP Kit Instructions for Use For selective enrichment of DNA fragments containing 5 hydroxymethylcytosine in a high throughput format using DNA isolated from various species This product is for research use only and is not intended for diagnostic use Table of Contents 1 Overview 2 Background 3 Components and Storage 4 Protocol 5 Troubleshooting 6 Related Products 1 Overview EpiSeeker 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 be used for various downstream applications including PCR hMeDIP PCR and microarray nhMeDI
6. ach wash step after adding Diluted EMH1 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 EMH1 is sufficient for at least two extra washes for each sample 19 Problem Cause Solution No difference in signal intensity between negative control and positive control Too many PCR cycles Plateau phase of amplification caused by over increased number of PCR cycles in endpoint PCR may mask the difference in signal intensity between negative control and positive control Decreasing the number of PCR cycles ex 32 35 cycles to keep amplification 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 generated from positive control only PCR conditions are not optimized Make sure the PCR conditions include correct and appropriate temperature cycles and solutions For further technical questions please do not hesitate to contact us by email technical abcam com or phone select contact us on www abcam com for the phone number for your region 20 6 Related Products EpiSeeker methylated DNA Immunoprecipitation MeDIP Kit DNA ab117133 EpiSeeker methylated DNA Immunoprecipitation MeDIP Kit ab117135 EpiSeeker methylated DN
7. e of 200 600 bp Oversized DNA fragments may reduce targeted DNA capturing via antibody and undersized DNA fragments may decrease PCR efficiency Incorrect temperature and or insufficient time during DNA release Ensure the incubation time and temperature described in the protocol are followed correctly Little or no PCR products generated from samples 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 18 Problem Cause Solution Little or no PCR products generated from samples 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 e
8. ed hMeDIP conditions e Compatible with various downstream analysis workflows including hMeDIP PCR and hMeDIP chip 3 Components and Storage A Kit Components Item Quantity Quantity Quantity 24 tests 48 tests 96 tests EMH1 10X Wash Buffer 5 mL 10 mL 20 mL EMH2 Antibody Buffer 4mL 8 mL 16 mL EMH3 hMeDIP Solution 3 mL 6 mL 12 mL EMH4 DNA Release Buffer 7 mL 14 mL 28 mL EMH5 Non Immune IgG 0 6 mg ml 10 uL 20 uL 40 uL EMH6 5 hmC Antibody 0 6 mg ml 25 uL 50 uL 100 uL EMH7 Control DNA 500 ng ml 5 uL 10 uL 20 uL EMH8 Proteinase K 10 mg ml 28 uL 56 uL 112 uL EMHg9 Control Primer Forward 20 uM 5 uL 10 uL 20 uL EMH10 Control Primer Reverse 20 uM 5 ul 10 uL 20 uL 8 Well Assay Strips With Frame 3 6 12 Adhesive 8 Well Strip Film 3 6 12 Spin the solution down to the bottom prior to use 10 Additional Materials Required e Variable temperature waterbath or incubator oven e Thermalcycler with 48 or 96 well block Sonication device e Orbital shaker e Adjustable pipette and multiple channel pipette e Aerosol resistant pipette tips e Parafilm M e 0 2 mlor 0 5 ml PCR vials Storage e Store EMH7 at 20 C away from light e Store EMH1 EMH5 EMH6 EMH8 EMH9 EMH10 and 8 Well Assay Strips at 4 C away from light e Store remaining components at room temperature away from light e All components of the kit are stable for 6 months from the date of shipment when stored pro
9. ed with this kit can be used for various downstream applications including PCR hMeDIP PCR and microarray nhMeDIP chip 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 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 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 The kit also includes control PCR primers that can be used for verifying the enrichment efficiency of hydroxymethylated control DNA The 5 hydroxymethylcytosine rabbit polyclonal antibody used in this kit is highly specific against hydroxymethylated DNA fragments and is not cross reactive to methylated and unmethylated DNA fragments 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 2 Background DNA methylation occurs by the covalent addition of a methyl group at the 5 carbo
10. n 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 a major 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 HH HH Ht ry ey rr ox ow of W c 5 C 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 P 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 particularly 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
11. perly Note Check if EMH1 contains salt precipitates before use If so briefly warm at room temperature or 37 C and shake the buffer until salts are re dissolved 11 4 Protocol Protocol Summary DNA shearing hmeDNA antibody immunoprecipitation Release of hmeDNA from hmeDNA Ab complex Elution of hmeDNA PCR microarray or similar downstream application A Starting Materials e Input DNA Amount DNA amount can range from 100 ng 1 ug per reaction An optimal amount is 500 ng per reaction e DNA Isolation You can use your method of choice for DNA isolation e DNA Storage Isolated genomic DNA can be stored at 4 C short term or 20 C long term until use 12 B Preparation of 1x Wash Buffer 1 Prepare Diluted EMH1 1X Wash Buffer Add 5 ml of EMH1 10X Wash Buffer to 45 ml of distilled water pH 7 2 7 5 This Diluted EMH1 1X Wash Buffer can now be stored at 4 C for up to six months C Preparation of Antibody Coated Wells 1 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 2 Add 100 ul of EMH2 to each well and then add the following antibodies 1 ul of EMH5 to the negative control well 1 ul of EMH6 to the sample wells and 1 ul of EMH6 to the positive control wells 3 Cover the wells with parafilm M and incubate at room tempera
12. ture for 60 min Meanwhile prepare fragmented DNA as described in the next step D Shearing of Genomic DNA 1 For the best results DNA should be fragmented by a suitable sonication method 13 a 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 microtip probe followed by a 30 40 sec rest period on ice between each pulse b Waterbath based__Sonication Follow the manufactuerer s user manual for DNA shearing at a size range of 200 600 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 with ethidium bromide Visualize it under ultraviolet light E Preparation of hMeDIP Reaction 1 Remove EMH2 from the wells and wash the wells two times with 200 ul of Diluted EMH1 each time 2 Dilute the EMH7 Control DNA to 50 ng ml 50 pg ul by adding 1 ul of EMH7 to 9 ul of EMH3 hMeDIP Solution and dilute your sample DNA with EMH3 hMeDIP Solution to 10 pg ml 10 ng ul Setup the hMeDIP reactions by adding the appropriate reagents to each corresponding well according to the following chart 14 Reagents Sample Positive Negative Negative well Control Control Control Well Well Well for for Control Sample DNA EMH3 50 uL 99 uL 50 uL 99 uL Sample DNA 50 uL N A 5
13. 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 an innovative method has been developed to capture DNA fragments containing 5 hmC and this method has been incorporated into the EpiSeeker 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 strip well microplate format 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 e Highly efficient enrichment ratio of positive negative control gt 1000 e Low DNA input requirement of as low as 0 1 ug per reaction e High reproducibility through pre optimiz
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