ChromaFlash™ One-Step Magnetic ChIP Kit
Contents
1. ChromaFlash One Step Magnetic ChIP Kit Base Catalog P 2026 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The ChromaFlash One Step Magnetic ChIP Kit is suitable for selective enrichment of a chromatin fraction containing specific DNA sequences in a high throughput format using chromatin isolated from various species particularly mammals Chromatin can be isolated by using your own successful method or for your convenience and the best results with Epigentek s ChromaFlash Chromatin Extraction Kit Cat P 2001 for mammals optimized for use with this product The target protein bound DNA prepared with the ChromaFlash One Step Magnetic ChIP Kit can be used for various downstream applications including PCR ChIP PCR microarrays ChIP chip and sequencing ChIP seq Use of EpiSonic Sonication The ChromaFlash One Step Magnetic ChIP Kit is optimized for use with the EpiSonic Multi Functional Bioprocessor 1000 Cat EQC 1000 in order to speed up the ChIP process and increase enrichment efficiency For use of this kit without the EpiSonic please see Standalone Protocol Input Amount of Chromatin The amount of chromatin for each reaction can be 0 1 pg about 1 x 104 cells to 15 ug about 1 5 x 10 cells For an optimal reaction the input chromatin amount should be 5 to 10 pg about 0 5 to 1 x 10 cells as enrichment of target proteins to genome loci varies and some of the target proteins are of2. 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 12 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 05 29 Epigentek Group Inc All rights reserved Products are for research use only P 2026 Com ponam Size pl Final Concentration C E E Total Voume fow ooo 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 95 C 10 sec Cycling 55 C 10 sec 40 72 C 8 sec Final Extension 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 ollgG CT Sample CT x 100 For example if CT for IgG is 38 and the sample is 34 then FE 2 x 100 1600 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
3. 2 3 min and then remove it 2 Add an additional one to two washes The provided volume of Diluted CH1 is sufficientfor 4 extra washes foreach sample Decrease the number of PCR cycles i e 32 35 cycles to keep ampification atthe exponential phase will reduce high background in endpoint PCR and allow differences in amplification to be seen Realtime PCR is another choice in such cases Increase the antibody amountand use ChIP grade antibodies validated for use in ChIP Confirm species specificity of primers Primers should be designed to cover a shortsequence region 70 150 bp for more efficientand precise amplification of target DNA region binding sites ofthe protein of interest 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 Web www epigentek com Printed 2014 05 29 Epigentek Group Inc All rights reserved Products are for research use only P 2026 STANDALONE PROTOCOL This protocol is intended for use without the EpiSonic Multi Functional Bioprocessor 1000 For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Input Chromatin Amount Chromatin amount can range from 0 1 g to 15 ug per reaction An optimal amount is 5 10 yg per reaction Chromatin Isolation You can use your method of choice for chromatin isolation Epigentek offers the ChromaFlash Chromatin
4. Caps 12 24 R 2 Ja JRT Magnetic Beads 220 ul 450 ul 4 C UserGuide 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 CH1 Non Immune IgG Anti RNA Polymerase II Proteinase K GAPDH Primer Forward GAPDH Primer Reverse and Magnetic Beads 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 CH1 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 Variable temperature waterbath or incubator oven Thermalcycler with 48 or 96 well block EpiSonic Multi Functional Bioprocessor 1000 Epigentek Cat EQC 1000 Incubator oven with variable temperature Orbital shaker Oo OF OF QO QO Magnetic stand 96 well format 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 Web www epigentek com Printed 2014 05 29 Epigentek Group Inc All rights reserved Products are for research use only P 2026 Adjustable pipette and multichannel pipette Aerosol resistant pipette tips 0
5. Extraction Kit Cat P 2001 for your convenience see Ordering Information section at the end of this manual Chromatin Storage Isolated chromatin can be stored at 20 C short term or 80 C long term until use 1 Chromatin Shearing If a probe based sonicator will be used the sonication settings need to be optimized by you For example DNA of 200 1000 bp size can be obtained by sonicating 3 4 pulses of 15 20 sec each at 40 output power using a Microson 1 8 inch Microtip probe followed by a 30 40 sec rest period on ice between each pulse If desired remove 10 ul of sheared chromatin for DNA purification and agarose gel analysis along with a DNA marker on a1 2 agarose gel stained with ethidium bromide and visualize it under ultraviolet light 2 Preparation of 1X Wash Buffer CH1 48 Reactions Kit Add 8 ml of CH1 10X Wash Buffer to 72 ml of distilled water pH 7 2 7 5 96 Reactions Kit Add 16 ml of CH1 10X Wash Buffer to 144 ml of distilled water pH 7 2 7 5 This Diluted CH1 1X Wash Buffer can now be stored at 4 C for up to six months 3 Preparation of One Step ChIP Reaction a Predetermine the number of PCR wells required for your experiment Cap the unused wells of the 96 Well PCR Plate with the supplied 8 Well Strips Caps before continuing further to avoid any possible contamination Plate can also be saved for later use Setup the ChIP reactions by adding the reagents to each well according to
6. IgG Immunoprecipitated DNA is then cleaned released and eluted Eluted DNA can be used for various downstream applications such as ChIP PCR ChIP on chip and ChIP seq 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 05 29 Epigentek Group Inc All rights reserved Products are for research use only P 2026 250 E Normal mouse IgG Clean and release DNA z 200 4 ERNA poIymerase I ng 150 2 a 100 Elute DNA 504 0 T iar 1 MLH1 Downstream analysis PCR microarray Fig 1 The data above shows the analy sis of enci enrichment of RNA poly merase II in GAPDH and oops ng etc MLH1 promoters by the ChromaFlash One Step Magnetic ChIP Kit with chromatin extract prepared from formaldehy de fixed colon cancer cells Captured DNA was used for analy zing levels of RNA Schematic procedure of the ChromaFlash poly merase II enriched in the GAPDH and MLH1 One Step Magnetic ChIP Kit promoters ASSAY PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Input Chromatin Amount Chromatin amount can range from 0 1 g to 15 pg per reaction An optimal amount is 5 10 yg per reaction Chromatin Isolation You can use your method of choice for chromatin isolation Epigentek offers the ChromaFlash Chrom
7. for use or storage at 20 C For real time PCR analysis we recommend the use of 1 2 ul of eluted DNA ina 20 ul PCR reaction If input DNA will be used it should be diluted 10 fold before adding to PCR reaction Control primers 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 05 29 Epigentek Group Inc All rights reserved Products are for research use only P 2026 110 bp for human cells included in the kit can be used as a positive control For end point PCR the number of PCR cycles may need to be optimized for better PCR results In general the amplification difference between normal IgG control and positive control may vary from 3 to 8 cycles depending on experimental conditions For ChIP chip or ChIP seq additional DNA clean up and concentration steps may be needed For your convenience Epigentek offers a DNA Concentrator Kit Cat P 1006 for DNA clean up and concentration TROUBLESHOOTING Little or NoPCR Products Generated from both Sample and Positive Control Wells Poor chromatin quality due to insufficientamountofcells or Poor enrichmentwith antibody some antibodies used in ChIP might not efficiently recognize fixed protein Inappropriate DNA fragmenting condition Incorrect temperature and or insufficienttime during DNA release Improper PCR condition
8. low abundance Starting Materials Starting materials can include various tissue or cell samples such as cells from flask or microplate cultured cells fresh and frozen tissues etc Antibodies Antibodies should be ChIP or IP grade as to recognize fixed and native proteins that are bound to DNA or other proteins If you are using antibodies which have not been validated for ChIP then appropriate control antibodies such as RNA Polymerase II Cat A 2032 should be used to demonstrate that the antibody and chromatin are suitable for ChIP Internal Control Both negative and positive DNA controls are provided in this kit 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 05 29 Epigentek Group Inc All rights reserved Products are for research use only P 2026 KIT CONTENTS Component CH1 10X Wash Buffer CH3 DNA Release Buffer RT Non Immune IgG 1 mg ml 4 Anti RNA Polymerase II 1 mg ml 4 Proteinase K 10 mg ml 4 GAPDH Primer Forward 20 uM 4 GAPDH Primer Reverse 20 uM 4 8 Well Strip
9. 20 ul PCR reaction If input DNA will be used it should be diluted 10 fold before adding to PCR reaction Control primers 110 bp for human cells included in the kit can be used as a positive control For end point PCR the number of PCR cycles may need to be optimized for better PCR results In general the amplification difference between normal IgG control and positive control may vary from 3 to 8 cycles depending on experimental conditions For ChIP chip or ChIP seq additional DNA clean up and concentration steps may be required For your convenience Epigentek offers a DNA Concentrator Kit Cat P 1006 for DNA clean up and concentration 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 Kit Cat P 1029 which is optimized for fast qPCR reactions As an example the protocol is presented below Prepare the PCR Reactions Thaw all reaction components including master mix DNA RNA free water primer solution and DNA template Mix well by vortexing briefly Keep components on ice while in use and return to 20 C immediately following use Add components into each well according to the following
10. 2ml or 0 5 ml PCR tubes Oo OF 0 Antibodies of interest OPTIONAL MATERIALS O Microplate centrifuge O Rolling shaker GENERAL PRODUCT INFORMATION Quality Control Each lot of the ChromaFlash One Step Magnetic ChIP 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 ChromaFlash One Step Magnetic ChIP Kit is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The ChromaFlash One Step Magnetic ChIP Kit and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Protein DNA i
11. atin Extraction Kit Cat P 2001 for your convenience Chromatin Storage Isolated chromatin can be stored at 20 C or 80 C long term until use 1 Preparation of 1X Wash Buffer CH1 48 Reactions Kit Add 8 ml of CH1 10X Wash Buffer to 72 ml of distilled water pH 7 2 7 5 96 Reactions Kit Add 16 ml of CH1 10X Wash Buffer to 144 ml of distilled water pH 7 2 7 5 This Diluted CH1 1X Wash Buffer can now be stored at 4 C for up to six months 2 Preparation of One Step ChIP Reaction 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 Web www epigentek com Printed 2014 05 29 Epigentek Group Inc All rights reserved Products are for research use only P 2026 a Predetermine the number of PCR wells required for your experiment Cap the unused wells of the 96 Well PCR Plate with the supplied 8 Well Strips Caps before continuing further to avoid any possible contamination b Setup the one step ChIP reactions by adding the reagents to each well according to the following Reagents Sample Positive Control Negative Control CH2 ChIP Buffer 55 75ul 55 75 ul 55 75 ul 20 40ul 20 40 pl 20 40 ul Your Antibodies O52ul Jo 0 RNAPolymerasell o f 0 8 ul Non mmuneiga 0 Jo Total Volume 1009 100p room Note The final amount of each component should be a chromatin 5 10 ug vell b antibodies of interest 0 8 ug well c RNA Polyme
12. e involves an excessive amount of steps inconsistency and sub optimized chromatin shearing These flaws result in inconvenience low throughput processing and less enrichment efficiency Epigentek first addressed these issues in 2005 by reducing the entire ChIP procedure to 5 hours in addition to improving upon performance and efficiency and now further refines its ChIP expertise with the ChromaFlash technology Because the major features of next generation sequencing and microarrays are their rapidness and high throughput capabilities these technologies are becoming major players in massive protein DNA analysis To be compatible with these new technologies rapid and massive generation of target protein bound DNA is critically required To meet this requirement Epigentek further developed a new ChIP technology called ChromaFlash and incorporated it into its ChromaFlash One Step Magnetic ChIP Kit With this kit and utilizing the EpiSonic Multi Functional Bioprocessor 1000 chromatin shearing and immunoprecipitation can be simultaneously processed which greatly advances ChIP to the highest speeds in a high throughput format with higher efficiency The ChromaFlash One Step Magnetic ChIP Kit has the following advantages e The fastest and most convenient ChIP method The entire procedure from intact chromatin sample to ready for use DNA is less than 60 minutes with the actual handling time being less than 10 minutes due to simultane
13. for research use only Page 8 Printed 2014 05 29 P 2026 Inproper sample storage No Differencein Signal Insufficient washing Intensity Between Negative and Positive Control Wells Too many PCR cycles Pleateu phase ofamplification caused by over increased number of PCR cycles in endpoint PCR may mask the difference of signal intensity between negative contol and positive control Little or No PCR Poor enrichmentwith antibody Products some antibodies used in ChIP Generated From Sample mightnot efficiently recognize Wells Only fixed protein PCR primers are notoptimized correctly mixed If using a PCR commercial kit check if it is suitable for your PCR Confirm species specificity of primers Primers should be designed to cover a shortsequence region 70 150 bp for more efficientand precise amplification of target DNA region 9 binding sites ofthe protein of interest Chromatin sample should be stored at 80 C for no longer than 6 months preferably less than 3 months Avoid repeated freeze thaw cycles DNA samples should be stored at 20 C for no longer than 6 months preferably less than 3 months Check if washing recommendations at each step is performed according to the protocol If the signal intensityin the negative control is still high washing stringencycan be increased in the following ways 1 Increase wash time ateach wash step after adding diluted CH1 leave it in the tubes wells for
14. 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 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 13 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 05 29 Epigentek Group Inc All rights reserved Products are for research use only P 2026 RELATED PRODUCTS ChIP Reaction P 2025 ChromaFlash One Step ChIP Kit Chromatin Preparation and Cleanup P 2001 ChromaFlash Chromatin Extraction Kit P 1006 DNA Concentrator Kit Sonication Instruments EQC 1000 EpiSonic Multi Functional Bioprocessor 1000 PCR Analysis P 1029 EpiQuik Quantitative PCR 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 Epigentek Group Inc All rights reserved Products are for research use only Page 14 Printed 2014 05 29 P 2026
15. l four times with 180 ul of the Diluted CH1 each time Each wash can be performed as follows Remove the plate from the magnetic stand after solution has been removed and add the Diluted CH1 to the reaction wells Resuspend the beads by gently pipetting up and down several times Ensure the pellet is completely resuspended and beads are not clinging to the pipette tips after pipetting Place the plate back to the magnetic stand for 1 2 min to pellet the beads and then remove and discard the solution from each reaction well Wash each reaction well one time with 180 ul of CH3 with the same wash procedure as above using CH3 instead of Diluted CH1 Remove and discard the CH3 solution from each well Remove the plate from the magnetic stand 4 Reversal of Cross Links Release and Elution of DNA a b Prepare the CH3 PK solution by adding 1 ul of Proteinase K to each 39 ul of CH3 and mix Add 40 ul of the CH3 PK solution to each reaction well then cover with new strip caps Incubate the wells at 65 C for 15 min and then 95 C for 5 min in a thermal cycler Remove the plate to room temperatue If liquid is collected on the inside of the caps briefly spin the liquid down to the bottom Place the plate on the magnetic stand 96 well format for 1 2 min to pellet the beads to the sides of the tubes wells Carefully transfer the supernatant from each reaction well to new 0 2 or 0 5 ml PCR tubes Cap the PCR tubes DNA is now ready
16. nteraction plays a critical role for cellular functions such as signal transduction gene transcription chromosome segregation DNA replication and recombination and epigenetic silencing Identifying the genetic targets of DNA binding proteins and knowing the mechanisms of protein DNA interaction is important for understanding cellular process Chromatin immunoprecipitation ChIP offers an advantageous tool for studying protein DNA interactions It allows for the detection that a specific protein binds to the specific sequences of a gene in living cells by PCR ChIP PCR microarrays ChIP chip or sequencing ChIP seq For Page 3 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 05 29 Epigentek Group Inc All rights reserved Products are for research use only P 2026 example measurement of the amount of methylated histone H3 at lysine 9 meH3 K9 associated with a specific gene promoter region under various conditions can be achieved through a ChIP PCR assay while the recruitment of meH3 K9 to the promoters on a genome wide scale can be detected by ChIP chip In particular ChIP with antibodies directly against various transcriptional factors is widely demanded However currently used ChIP methods have several drawbacks of which the most critical weakness is lengthy procedures often taking up to 3 days to finish the procedures Additionally the labor intensive procedur
17. ous processing of chromatin shearing and immunoprecipitat ion One Step ChIP e 96 vell plate format makes the assay flexible Either a manual with one single reaction each time or b high throughput with 96 reactions each time e Highly efficient enrichment Enrichment ratio of positive to negative control gt 120 and an extremely low number of cells required as low as 10 000 cells per ChIP reaction e High reproducibility Pre optimized ChIP conditions and with the EpiSonic Multi Functional Bioprocessor 1000 digitally acoustic controlled reaction processing in sealed vials make the ChIP procedure consistent e Wide downstream analysis compatibility Compatible with various downstream analysis workflows including ChIP PCR ChIP on chip and ChIP seq PRINCIPLE amp PROCEDURE The ChromaFlash One Step Magnetic ChIP Kit contains all necessary reagents required for carrying out a successful chromatin immunoprecipitation directly from chromatin extracts isolated from mammalian cells or tissues This kit includes a positive control antibody RNA polymerase Il a negative control non immune IgG and GAPDH primers that can be used as a positive control to demonstrate the efficacy of the kit reagents and protocol RNA polymerase Il is considered to be enriched in the GAPDH gene promoter that is expected to be undergoing transcription in most growing mammalian cells and can be immunoprecipitated by RNA polymerase II but not by non immune
18. pette tips after pipetting Place the plate back to the magnetic stand for 1 2 min to pellet the beads and then remove and discard the solution from each reaction well Wash each reaction well one time with 180 ul of CH3 with the same wash procedure as above using CH3 instead of Diluted CH1 Remove and discard the CH3 solution from each well Remove the plate from the magnetic stand 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 05 29 Epigentek Group Inc All rights reserved Products are for research use only P 2026 5 Reversal of Cross Links Release and Elution of DNA a Prepare CH3 PK solution by add 1 ul of Proteinase K to each 39 ul of CH3 and mix b Add 40 ul of the CH3 PK solution to each well then cover with new strip caps c Incubate the wells at 65 C for 15 min and then 95 C for 5 min in a thermal cycler d Remove the plate to room temperatue If liquid is collected on the inside of the caps briefly spin the liquid down to the bottom e Place the plate on the magnetic stand 96 well format for 1 2 min to pellet the beads to the sides of the tubes wells f Carefully transfer the supernatant from each reaction well to new 0 2 or 0 5 ml PCR tubes Cap the PCR tubes DNA is now ready for use or storage at 20 C For real time PCR analysis we recommend the use of 1 2 ul of eluted DNA in a
19. rase II 0 8 ug well and d non immune IgG 0 8 ug well Freshly prepared chromatin can be used directly for the reaction Frozen chromatin samples should be thawed quickly at RT and then placed on ice before use Store remaining chromatin samples at 20 T or at 80 if they will not be used within 8 hours The amounts of the positive control and negative control are sufficient for matched use with samples if two antibodies are used for each sample or one antibody is used for two of the same samples If using one antibody of interest for each sample with matched use of the positive and negative control extra RNA polymerase II and non immune IgG required can be separately obtained from Epigentek Input DNA control is only used for estimating the enrichment efficiency of ChIP and is generally not necessary as the positive and negative control can be used for estimating the same objective more accurately If you would like to include the input DNA control the following steps can be carried out 1 add 10 ul of each chromatin sample to a 0 2 ml PCR tube followed by adding 88 ul of CH3 and 2 5 ul of Proteinase K 2 incubate the input DNA control at 65 C for 15 min then incubate at 95 for 10 min and 3 spin the solution down to the bottom Inout DNA is ready for PCR or storage at 20 C c Cap the reaction wells with the 8 Well Strip Caps and place the 96 Well PCR Plate to the platform of the sample processing horn of the EpiSonic Multi Func
20. ry as the positive and negative control can be used for estimating the same objective more accurately If you would like to include the input DNA control the following steps can be carried out 1 add 10 pl of each chromatin sample to a 0 2 ml PCR tube followed by adding 88 ul of CH3 and 2 5 ul of Proteinase K 2 incubate the input DNA control at 65 TC for 15 min followed by incubating at 95 for 10 min and 3 spin the solution down to the bottom Input DNA is ready for PCR or storage at 20 T Cap the reaction wells with the 8 Well Strip Caps and place the 96 Well PCR Plate on a rolling shaker for 120 min Tape the plate to the rolling shaker securely Remove the plate from the rolling shaker If liquid is found in the caps briefly spin the plate down using a microplate centrifuge 4 Washing of the Reaction Wells Carefully remove the caps from the reaction wells Place the plate on a magnetic stand 96 well format for 1 2 min in order to pellet the beads to the sides of the wells Carefully remove and discard the solution from each reaction well Wash each reaction well four times with 180 ul of the Diluted CH1 each time Each wash can be performed as follows Remove the plate from the magnetic stand after solution has been removed and add the Diluted CH1 to the reaction wells Resuspend the beads by gently pipetting up and down several times Ensure the pellet is completely resuspended and beads are not clinging to the pi
21. s including improper PCR programming PCR reaction solutions and or primers insufficient or over cross linking To obtain an optimal amountof chromatin per ChIP reaction should be 5 10 ug about0 5 1 x 10 cells The minimum amountof chromatin is 0 05 ug 5 000 cells Appropriate chromatin cross linking is also required Insufficient or over crosslinking will cause DNAloss or increased background During cross linking step of chromatin preparation ensure thatthe cross linking time is within 10 15 min the concentration of formaldehyde is 1 as the final concentration and or quench solution is 0 125 M glycine Increase the antibody amountand use ChIP grade antibodies validated for use in ChIP If chromatinis from specific cellAissue types suchas plant or is differently fixed the processing program mustbe modified see EpiSonic manual to optimize the processing results If using a probe based sonicator shearing conditions should also be optimized to allow DNA fragmentsize to be between 200 1000 bp Ensure the incubation times and temperatures described in the protocol are followed correctly Ensure the PCR is properly programmed If using ahomebrew PCR reaction solution check if each componentis 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
22. the following chart Reagents Sample Positive Control Negative Control CH2 ChIP Buffer 55 75ul 55 75 ul 55 75 ul 20 404 Your Anibodies os2w o fo RNA Polymerase Il 0 8 ul 0 Fnonimmunetyg 0 To To 4 Total Volume 100m 1004 TOO 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 10 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 05 29 Epigentek Group Inc All rights reserved Products are for research use only P 2026 Note The final amount of each component should be a chromatin 5 10 ug well b antibodies of interest 0 8 ug well c RNA Polymerase II 0 8 ug well and d non immune IgG 0 8 ug well Freshly prepared chromatin can be directly used for the reaction Frozen chromatin samples should be thawed quickly at RT and then placed on ice before use Store remaining chromatin samples at 20 or at 80 if they wil be not used within 8 hours The amounts of the positive control and negative control are sufficient for matched use wth samples if two antibodies used for each sample or one antibody is used for two of the same samples If using one antibody of interest for each sample wth matched use of the positive and negative control extra RNA polymerase II and non immune IgG required can be separately obtained from Epigentek Input DNA control is only used for estimating the enrichment efficiency of ChIP and is generally not necessa
23. tional Bioprocessor 1000 d Start up the EpiSonic by following the standard EpiSonic operation instructions Slowly add ice cold filtered water into the sample processing horn so that your sample contents in the strip wells are submerged just below the water level e Set the EpiSonic to the following program using the power output chart under Optimization Suggestions in its manual as a reference 20 sec Pulse ON at 120W 20 sec Pulse OFF Total 20 duty cycles Total ON time should be 3 40 10 min ON at 40W 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Pag e6 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 05 29 Epigentek Group Inc All rights reserved Products are for research use only P 2026 The above program can be modified based on the amount of input chromatin and the cell tissue type from which the chromatin is prepared from For processing ChIP without using the EpiSonic see Standalone Protocol Gently remove the 96 Well PCR Plate without tilting If liquid is found in the caps briefly spin the plate using a microplate centrifuge 3 Washing of the Reaction Wells f g Carefully remove the caps from the reaction wells Place the plate on a magnetic stand 96 well format for 1 2 min in order to pellet the beads to the sides of the wells Carefully remove and discard the solution from each reaction well Wash each reaction wel
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