ChromaFlash ™ Chromatin Isolation & Shearing Kit
Contents
1. ChromaFlash Chromatin Isolation amp Shearing Kit Base Catalog P 2023 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The ChromaFlash Chromatin Isolation amp Shearing Kit is suitable for isolating and shearing chromatin from mammalian cells or tissues in a simple and rapid format Chromatin sheared with this kit can be used in a variety of chromatin immunoprecipitation methods The sheared chromatin can also be used in other chromatin related applications such as in vitro protein DNA binding assays Starting Material and Input amount Starting materials can include various tissue or cell samples such as cells from flask or microplate cultured cells fresh and frozen tissues etc The amount of cells and tissues for each preparation can be 2 x 10 to 1 x 10 cells and 10 mg to 400 mg respectively For optimal preparation the input amount should be 2 to 5 x 10 cells or 100 to 200 mg tissues A total of 100 standard extractions use 2 X10 cells or 100 mg of tissue per extraction can be performed with this kit The yield of sheared chromatin is approximately 3 ug per 10 cells or per 50 mg tissues Precautions To avoid cross contamination carefully pipette the sample or solution into the tube vials 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 Blvd2. Ste 122 Farmingdale NY 11735 Page 1 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 2023 KIT CONTENTS Component 100 Preparations Storage P 2023 100 Upon Receipt 10X Lysis Buffer 11 ml RT Extraction Buffer 22 ml RT Chromatin Buffer 22 ml RT Protease Inhibitor Cocktails 1000X 110 ul 4 C User Guide 1 RT Spin the solution down to the bottom prior to use SHIPPING amp STORAGE The kit is shipped on frozen ice packs at 4 C Upon receipt 1 Protease Inhibitor cocktails at 4 C 2 Store remaining components at room temperature All components of the kit are stable for 6 months from the date of shipment when stored properly Note Check if any buffers contain salt precipitates before use If so shake the buffer until the salts are re dissolved MATERIALS REQUIRED BUT NOT SUPPLIED o o O oO o o o o O Oo oO o Sonicator Vortex mixer Dounce homogenizer Centrifuge including desktop centrifuge up to 14 000 rpm Pipettes and pipette tips 1 5 ml microcentrifuge tubes 15 ml conical tube Cells or tissues Cell culture medium 37 formaldehyde if cross linked 1 25 M Glycine solution if cross linked 1X PBS 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail i
3. the correct volume and in the correct order based on the sample amount Check for sample lysis under microscope after the tissue cell lysis step Ensure that the cell or tissue species 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 Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 2023 are compatible with this extraction procedure Lysis or extraction reagents have expired Expired reagents may cause inefficient extraction Ensure that the kit has not exceeded the expiration date of the kit Standard shelf life when stored properly is 6 months from date of receipt Incorrect temperature and or insufficient incubation time during extraction Ensure the incubation time and temperature described in the protocol are followed correctly Degradation of chromatin Improper storage of chromatin Chromatin sample should be stored at 80 C 3 6 months Avoid multiple freeze thaw cycles There is a cloudy layer in the sheared chromatin solution Lipid and glycogen are not completely removed during chromatin extaction Avoid this layer when supernatant is removed Chromatin is not efficiently sheared Shearing condition is not optimized especially when probe based sonicator is used To obtain desi
4. e 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 Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 09 22 P 2023 Prepare cross link solution by adding formaldehyde to cell culture medium with a final concentration of 1 e g add 270 ul of 37 formaldehyde to 10 ml of culture medium Add 1 ml of cross link solution for every 50 mg tissues Incubate at room temperature for 15 20 min on a rocking platform Add 1 ml of 1 25 M glycine for every 9 ml of cross link solution Mix and centrifuge at 800 rpm for 5 min Discard the supernatant Wash cells with 10 ml of ice cold PBS once by centrifugation at 800 rpm for 5 min Discard the supernatant Transfer tissue pieces to a Dounce homogenizer Add 0 5 ml Working Lysis Buffer for every 50 mg tissues Disaggregate tissue pieces by 20 40 strokes Transfer homogenized mixture to a 15 ml conical tube and centrifuge at 3000 rpm for 5 min at 4 C If total mixture volume is less than 2 ml transfer mixture to a 2 ml vial and centrifuge at 5000 rpm for 5 min at 4 C Then go directly to Step 3f 3 Cell Lysis and Chromatin Extraction h Add 1 ml of 1 25 M glycine for every 9 ml of cross link solution Mix and centrifuge at 1000 rpm for 5 min Remove medium and wash cells once with 10 ml of ice cold PBS by centrifuging at 1000 rpm for 5 min Add Working Lysis Buffer to re susp
5. end the cell pellet 200 l 1x10 cells and incubate on ice for 10 min Vortex vigorously for 10 sec and centrifuge at 3000 rpm for 5 min Carefully remove supernatant Add Working Extraction Buffer to re suspend the chromatin pellet 100 l 1x10 cells 500 ul maximum for each vial Transfer the chromatin lysate to a 1 5 ml vial and incubate on ice for 10 min and vortex occasionally 4 Chromatin Shearing a Resuspend the chromatin lysate by vortexing b Shear chromatin with one of the following methods Waterbath Sonication 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 Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 09 22 P 2023 Epigentek EpiSonic 1100 Epigentek Cat No EQC 1100 Use 50 ul of chromatin lysate per 0 2 ml tube or per PCR plate well Shear 20 cycles 15 sceond On 30 second Off each at 170 190 watts Diagenode Bioruptor Use 200 ul of chromatin lysate per 1 5 ml vial Shear 3 runs of 10 cycles 30 second On 30 second Off each at high power setting Spin and vortex between each run Covaris S2 Instrument Use 130 ul of chromatin lysate per Covaris microTube Duty cycle 2 Intensity 3 Cycles per burst 200 Total processing time 10 min Probe based Sonication Use 300 ul of chromatin lysate per 1 5 ml tube As an example sonication can be carried
6. mine if a specific protein binds to the specific sequences of a gene in living cells by combining ChIP with PCR ChIP PCR microarray ChIP chip or sequencing ChIP Seq techniques For example the measurement of the amount of methylated histone H3 at lysine 9 meH3 K9Y associated with a specific gene promoter region under various conditions can be achieved through a ChIP PCR assay while recruitment of meH3 K9 to the promoters on a genome wide scale can be detected by ChIP chip In particular the ChIP method with specific antibodies directly against various transcriptional factors is widely demanded For performing ChIP chromatin or DNA protein complex in cells or tissues should first be isolated and sheared to desired size The ChromaFlash Chromatin Isolation amp Shearing Kit addresses the inconvenience and time consuming issues of existing chromatin preparation methods by introducing the following features e Fast procedure the entire procedure from cell tissue sample to ready to use sheared chromatin is less than 2 hours e Convenient and flexible the kit is suitable for preparing both native chromatin and cross linked chromatin from monolayer or suspension cells or from tissues e Chromatin shearing is compatible with various sonication platforms including waterbath based probe based sonicators and enzyme based methods PRINCIPLE amp PROCEDURE The ChromaFlash Chromatin Isolation amp Shearing Kit contains all reagents
7. natant Note For cells that are not cross linked go directly to Step 3d after Step 2c d Add 9 ml fresh cell culture medium containing formaldehyde with a final concentration of 1 i e add 270 ul of 37 formaldehyde to 10 ml of cell culture medium to cells e Incubate at room temperature 20 25 C for 10 min on a rocking platform 50 100 rpm For Suspension Cells a Collect cells treated or untreated into a 15 ml conical tube 2 x10 to 5x10 cells are required for each preparation Count cells in a hemocytometer b Centrifuge the cells at 1000 rpm for 5 min Discard the supernatant c Wash cells with 10 ml of PBS once by centrifugation at 1000 rpm for 5 min Discard the supernatant Note For cells that are not cross linked go directly to Step 3d after Step 2c d Add 9 ml fresh cell culture medium containing formaldehyde with a final concentration of 1 i e add 270 ul of 37 formaldehyde to 10 ml of cell culture medium to cells e Incubate at room temperature 20 25 C for 10 min on a rocking platform 50 100 rpm For Tissues a Put the tissue sample into a 60 or 100 mm plate Remove unwanted tissue such as fat and necrotic material from the sample b Weigh the sample and cut the sample into small pieces 1 2 mm with a scalpel or scissors Note For tissues that are not cross linked go directly to Step 2j after Step 2b c Transfer tissue pieces to a 15 ml conical tube 110 Bi County Blvd Ste 122 Farmingdal
8. nfo epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 2 Printed 2014 09 22 P 2023 O Distilled water GENERAL PRODUCT INFORMATION Quality Control Each lot of the ChromaFlash Chromatin Isolation amp Shearing 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 Chromatin Isolation amp Shearing Kit is for research use only and is not intended for diagnostic or therapeutic application A BRIEF OVERVIEW Chromatin immunoprecipitaton ChIP offers an advantageous tool for studying protein DNA interaction The experimenter can deter
9. out with a microtip attached to a Branson 450 sonifier set to 25 power output Sonicate 3 4 pulses of 10 15 seconds each followed by 30 40 seconds rest on ice between each pulse The conditions of cross linked DNA shearing can be optimized based on cells and sonicator equipment If desired remove 10 ul of sonicated cell lysate for agarose gel analysis along with a DNA marker on a 1 2 agarose gel Stain with Ethidium Bromide or other fluorescent dye for DNA and visualize it under ultraviolet light The length of sheared DNA should be between 200 1000 bp Note The isolated chromatin can also be sheared with various enzyme based methods The optimization of the shearing conditions for example enzyme concentration and incubation time is needed in order to use enzyme based methods Centrifuge at 12 000 rpm at 4 C for 10 min Transfer supernatant to a new vial Add Chromatin Buffer at a 1 1 ratio e g add 100 ul of Chromatin Buffer to 100 ul of supernatant The chromatin solution can now be used immediately or stored at 80 C after aliquoting appropriately until further use Avoid multiple freeze thaw cycles TROUBLESHOOTING Problem Possible Cause Suggestion Low yield of chromatin Insufficient amount of samples To obtain the best results the amount of samples should be 2 to 5 x 10 cells or 100 to 200 mg tissues per ChIP reaction Insufficient chromatin extraction Ensure that all reagents have been added with
10. red DNA size 200 1000 bps shear chromatin with a time course ex 5 10 15 20 pulses that generates suitably sized DNA for use in ChIP In general waterbath based sonicators such as the EpiSonic 1100 could give consistent and efficient shearing conditions RELATED PRODUCTS Chromatin Shearing and Cleanup P 1006 Sonication Instruments EpiSonic Multi Functional Bioprocessor 1100 EQC 1100 ChIP Reaction P 2025 P 2026 PCR Analysis P 1029 DNA Concentrator Kit ChromaFlash One Step ChIP Kit ChromaFlash One Step Magnetic ChIP kit EpiQuik Quantitative PCR Fast Kit ChIP Grade Antibodies For ChIP Grade Antibodies search chip grade at www epigentek com 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 2023
11. required for carrying out successful chromatin extraction and shearing directly from mammalian cells or tissues Cell 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 Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 2023 j bb co s g sce WB membranes of the sample with or without cross linking are broken down using the provided lysis buffer Chromatin or DNA protein complex is then extracted with the extraction buffer The extracted chromatin can then be sheared and stored at the appropriate temperature after being diluted in chromatin buffer g Start with cell or tissue sample with or without P cross linking Lyse sample with lysis ffer buffe Chromatin was extracted from six MDA 231cell samples 40 ul 8 ug of chromatin were sheared in a 0 2 ml tube using the EpiSonic 1100 for 20 cycles 15 sec On and 30 sec Off DNA was purified and then run on a gel Extract chromatin with extraction buffer 18 4 16 4 Sample 1 14 Sample 2 Pd O Sample 3 12 4 3 Sonicate to obtain 104 sheared chromatin 34 lt ae 4 4 2 uid Schematic procedure of the ChromaFlash o e i Chromatin Isolation Shearing Kit GAPDH MLH1 The sheared chromatin was used for ChIP qPCR analysis of RNA polymerase II enrichment in GAPDH and MLH1 promoters by using
12. the ChromaFlash One Step ChIP Kit P 2025 and the Methylamp MS qPCR Fast Kit P 1028 Assay Protocol For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Monolayer cells 2x10 to 1x10 cells per preparation Suspension cells 4x10 to 2x10 cells per preparation Tissues 10 mg to 400 mg per preparation 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 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 2023 1 Preparation of Working Buffers and Solutions a Prepare Working Lysis Buffer by adding 1 ml of 10X Lysis Buffer and 6 ul of Protease Inhibitor Cocktail to every 9 ml of distilled water Prepare Working Extraction Buffer by adding 1 ul of Protease Inhibitor Cocktail to every 1 ml of Extraction Buffer 2 Cell Collection and Cross Linking For Monolayer or Adherent Cells a Grow cells treated or untreated to 80 90 confluence on a 100 mm plate about 2x10 to 4x10 cells 2x10 cells are required for each standard preparation then trypsinize and collect them into a 15 ml conical tube Count the cells in a hemocytometer b Centrifuge the cells at 1000 rpm for 5 min Discard the supernatant c Wash cells with 10 ml of PBS once by centrifugation at 1000 rpm for 5 min Discard the super
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