ChromaFlash ™ One-Step ChIP Kit
Contents
1. e 96 well 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 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 kitincludes 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 IgG Immunoprecipitated DNA is then cleaned released and eluted Eluted DNA can be use2. ChromaFlash One Step ChIP Kit Base Catalog P 2025 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The ChromaFlash One Step 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 and Cat P 2022 for plants optimized for use with this product The target protein bound DNA prepared with the ChromaFlash One Step 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 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 of low abund
3. Generated from both insufficient or over cross linking 0 5 10 ug about 0 5 1 x 10 cells The Sample and Positive minimum amountofchromatinis 0 05 Control Wells ug 5 000 cells 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 11 14 Epigentek Group Inc All rights reserved Products are for research use only P2025 No Differencein Signal Poor enrichmentwith antibody some antibodies usedin ChIP might not efficiently recognize fixed protein Inappropriate DNA fragmenting condition Incorrect temperature and or insufficienttime during DNA release Improper PCR conditions including improper PCR programming PCR reaction solutions and or primers Inproper sample storage 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 timeis within 10 15 min the concentration of formaldehyde is 1 as the final concentration and or quench solution is 1 25 M glycine Increase the antibody amountand use ChIP grade antibodies validated for use in ChIP If chromatin is from specific cell tis sue types suchas plant or is differently fixed the processing program mustbe modified see EpiSonic manual to optimize the processing results If usin
4. 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 strip cap c Incubate the wells at 65 C for 15 20 min d Quickly transfer the DNA solution from each well to 0 2 ml strip PCR tubes Cap the PCR tubes e Incubate the PCR tubes containing DNA solution at 95 C for 5 10 min in a thermalcycler f Place the PCR tubes in room temperature If liquid is collected on the inside of the caps briefly spin the liquid down to the bottom 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 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 needed For your convenience Epigentek offers a DNA Concentrator Kit Cat P 1006 for DNA clean up and concentration TROUBLESHOOTING Little or NoPCR Poor chromatin quality due to To obtain an optimal amountof Products insufficient amountofcells or chromatin per ChIP reaction should be
5. 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 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 11 14 Epigentek Group Inc All rights reserved Products are for research use only P 2025 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 Component Size pl Final Concentration 50 p3 0 1 9 Total Voume fow OOo i 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 follows Cycle Step Gye 95 C 10sec Cycling 55 C 10 sec 40 72 C 8 sec Final Eviension Fold Enrichment Calculation Fold enrichment FE can be calculated by simply using a ratio of amplification efficiency of the ChIP sample over
6. 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 C if they will not be used within 8 hours The amounts of the positive control and negative control are sufficient for matched use wth 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 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 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 Input DNA is ready for PCR or storage at 20 Seal the wells with the Adhesive Covering Film and place the wells held on the Sonication Frame to the platform of the sample processing horn of the EpiSonic
7. Flash Chromatin Extraction Kit P 2023 ChromaFlash Chromatin Isolation and Shearing Kit P 1006 DNA Concentrator Kit Sonication Instruments EQC 1100 EpiSonic Multi Functional Bioprocessor 1100 PCR Analysis P 1029 EpiQuik Quantitative PCR Kit For ChIP Grade Antibodies search chip grade at ww epigentek com 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 Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 11 14 P 2025
8. Multi Functional Bioprocessor 1000 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 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 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 11 14 Epigentek Group Inc All rights reserved Products are for research use only P2025 For processing ChIP without using the EpiSonic see Standalone Protocol f Gently remove the Sonication Frame without tilting and carefully peel away the Adhesive Covering Film to avoid contamination between each well 3 Washing of the Reaction Wells a Carefully remove the solution and discard from each well b Wash each well with 200 ul of the Diluted CH1 each time for four times This can be done by simply pipetting Diluted CH1 in and out of the well c Wash each well with 200 ul of CH3 one time by pipetting CH3 in and out of the well 4 Reversal of Cross Links Release and Elution of DNA a Prepare the CH3 PK solution by adding 1 ul of
9. ance 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 Controls 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 201 fea 4 Epigentek Group Inc All rights reserved Products are for research use only P2025 KIT CONTENTS Cat P 2025 48 Cat P 2025 96 Upon Receipt CH1 10X Wash Buffer 4 CH2 ChIP Buffer Anti RNA Polymerase II 1 mg ml Proteinase K 10 mg ml 4 C GAPDH Primer Forward 20 uM 16 ul 4 C GAPDH Primer Reverse 20 pM 16 ul 8 Well Assa
10. can now be stored at 4 C for up to six months 3 Preparation of One Step ChIP Reaction 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 Setup the ChIP reactions by adding the reagents to each well according to the following chart Reagents Sample Positive Control Negative Control CH2 ChIP Buffer 50 60 pl 50 60 ul 50 60 ul 40 50 pl 40 50 ul 40 50 ul Your Antibodies 0 5 2 ul 0 RNA Polymerase 0 E NonimmunegG of o 084 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 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 e
11. com Printed 2014 11 14 Epigentek Group Inc All rights reserved Products are for research use only P 2025 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 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 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 simultaneous processing of chromatin shearing and immunoprecipitation One Step ChIP
12. d 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 11 14 Epigentek Group Inc All rights reserved Products are for research use only P 2025 Simultaneously shear i ini 150 and immunoprecipitate E Normal mouse IgG chromatin 130 5 O RNA polymerase II K 110 5 5 90 4 Clean and release DNA ww 704 Fi 2 a amp d 304 Elute DNA 10 5 H l a0 GAPDH MLH1 Downstream analysis Fig 1 The data abov e shows the analy sis of PCR microarray enrichment of RNA poly merase Il in GAPDH and sequencing etc MLH1 promoters by the ChromaFlash One Step ChIP Kit with chromatin extract prepared from formaldehy de fixed colon cancer cells Captured DNA was used for analy zing levels of RNA poly merase II enriched in the GAPDH and MLH1 promoters Schematic procedure of the ChromaFlash One Step ChIP Kit 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 Chromatin Extractio
13. e 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 strip cap c Incubate the wells at 65 C for 15 20 min d Quickly transfer the DNA solution from each well to 0 2 ml strip PCR tubes Cap the PCR tubes e Incubate the PCR tubes containing DNA solution at 95 C for 5 10 min in a thermalcycler f Place the PCR tubes in room temperature If liquid is collected on the inside of the caps briefly spin the liquid down to the bottom 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 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
14. g 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 correctly mixed If usingaPCR 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 binding sites of the 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 Insufficientwashing Intensity Between each stepis performed according to the 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 11 14 P 2025 Negative and Positive Control Wells Little or NoPCR Products Generated From Sample Wells Only Too many PCR cycles Pleateu phase ofamplification caused by over increased numbe
15. in Amount Chromatin amount can range from 0 1 g to 15 pg per reaction An optimal amount is 5 10 ug per reaction Chromatin Isolation You can use your method of choice for chromatin isolation Epigentek offers the ChromaFlash Chromatin Extraction Kit Cat P 2001 for your convenience Chromatin Storage Isolated chromatin can be stored at 20 C short term or 80 C long term until use 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 11 14 P 2025 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 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 If desired remove 10 ul of sheared chromatin for DNA purification and agarose gel analysis along with a DNA marker ona 1 2 agarose gel stained with ethidium bromide and visualize it under ultraviolet light 2 Preparation of 1X Wash Buffer CH1 48 Reactions Kit Add 10 ml of CH1 10X Wash Buffer to 90 ml of distilled water pH 7 2 7 5 96 Reactions Kit Add 20 ml of CH1 10X Wash Buffer to 180 ml of distilled water pH 7 2 7 5 This Diluted CH1 1X Wash Buffer
16. n Kit Cat P 2001 for your convenience Chromatin Storage Isolated chromatin can be stored at 20 C short term or 80 C long term until use 1 Preparation of 1X Wash Buffer CH1 48 Reactions Kit Add 10 ml of CH1 10X Wash Buffer to 90 ml of distilled water pH 7 2 7 5 96 Reactions Kit Add 20 ml of CH1 10X Wash Buffer to 180 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 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 11 14 Epigentek Group Inc All rights reserved Products are for research use only P2025 2 Preparation of One Step ChIP Reaction Predetermine the number of strip wells required for your experiment Carefully remove the strip wells you need from the plate frame and transfer them to the reverse side of the Sonication Frame upside down Unused strip wells can be placed back in the bag seal the bag tightly and store at 4 C b Setup the one step ChIP reactions by adding the reagents to each well according to the following Reagents he Sample x Positive Negative Control Control vor misoes osz o 01 Cana Papmaraset 0 oar o Non tmmuneigG o of osu 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 Polymerase II
17. or 0 5 ml PCR vials Antibodies of interest Oo OF 8 Qa OQ Orbital shaker for standalone protocol GENERAL PRODUCT INFORMATION Quality Control Each lot of the ChromaFlash One Step 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 ChIP Kit is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The ChromaFlash One Step ChIP Kit and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW Protein DNA interaction plays a critical role for cellular functions such as signal transduction gene transcription ch
18. r of PCR cycles in endpoint PCR may mask the difference of signal intensity between negative contol and positive control Poor enrichmentwith antibody some antibodies used in ChIP might not efficiently recognize fixed protein PCR primers are notoptimized 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 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 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 for each sample Decrease the number of PCR cycles i e 32 35 cycles to keep ampification at the exponential phase will reduce high background in endpoint PCR and allow differences in amplification to be seen Realtime PCR is another choice in suchcases 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 efficient and precise amplification of target DNA region binding sites of the protein of interest Input Chromat
19. romosome 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 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 procedure involves an excessive amount of steps inconsistency and sub optimized chromatin shearing These flaws result in inconvenience low throughput processing and less enrichment 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Pag e3 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek
20. stimating 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 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 20 110 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 m Web www epigentek com Printed 2014 11 14 Epigentek Group Inc All rights reserved Products are for research use only P 2025 C d Seal the wells with Adhesive Covering Film and incubate the wells at room temperature for 90 120 min on an orbital shaker 100 rpm Peel away the Adhesive Covering Film carefully to avoid contamination between each well 4 Washing of the Reaction Wells a b C Carefully remove the solution and discard from each well Wash each well with 200 ul of the Diluted CH1 each time for four times This can be done by simply pipetting Diluted CH1 in and out of the well Wash each well with 200 ul of the CH3 one time by pipetting CH3 in and out of the well 5 Reversal of Cross Links Releas
21. that of non immune IgG Amplification efficiency of Polymerase RNA II can be used as a positive control FE 2086 Sample D y 100 For example if CT for IgG is 38 and the sample is 34 then FE 208 30 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 in order to make 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Pag e 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 11 14 Epigentek Group Inc All rights reserved Products are for research use only P 2025 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 ChIP Reaction P 2026 ChromaFlash Magnetic One Step ChIP Kit Chromatin Preparation and Cleanup P 2001 Chroma
22. y Strips With 1 Frame 8 Well Strip Caps 1 96 Well PCR Plate 1 Adhesive Covering Film 1 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 GADPH Primer Reverse and 8 Well Assay Strips With 1 Frame 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 Variable temperature waterbath or incubator oven Thermalcycler with 48 or 96 well block EpiSonic Multi Functional Bioprocessor 1000 Epigentek Cat EQC 1000 Oo mgm 0 Incubator oven with variable temperature 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 11 14 Epigentek Group Inc All rights reserved Products are for research use only P 2025 Adjustable pipette and multiple channel pipette Aerosol resistant pipette tips 0 2 ml
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