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Circulating Cell-Free DNA Isolation Kit
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1. Magnetic stands suitable for 1 7 ml microtube 0 2 ml PCR tube and 96 well plate Page 2 Printed 2015 06 16 P 1064 GENERAL PRODUCT INFORMATION Quality Control Each lot of EpiQuik Circulating Cell Free DNA Isolation Kit is tested against predetermined specifications to ensure consistent product quality Epigentek guarantees the performance of all products in the manner described in our product instructions Product Warranty If this product does not meet your expectations simply contact our technical support unit or your regional distributor We also encourage you to contact us if you have any suggestions about product performance or new applications and techniques Safety Suitable lab coat disposable gloves and proper eye protection are required when working with this product Product Updates Epigentek reserves the right to change or modify any product to enhance its performance and design The information in this User Guide is subject to change at any time without notice Thus only use the User Guide that was supplied with the kit when using that kit Usage Limitation The EpiQuik Circulating Cell Free DNA Isolation Kit is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The EpiQuik Circulating Cell Free DNA Isolation Kit and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW Genetic and epigenetic analysis of circulating
2. EpiQuik Circulating Cell Free DNA Isolation Kit Base Catalog P 1064 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiQuik Circulating Cell Free DNA Isolation Kit utilizes magnetic beads based size fractionation technology to isolate circulating cell free DNA ccfDNA from mono and di nucleosomal complexes in plasma serum samples The isolated ccfDNA can be directly used for real time PCR and DNA library preparation suitable for next generation sequencing Starting Material and Input Amount Plasma or serum from various species Input amount can be from 0 1 1 ml however the standard input amount is 0 5 ml per sample The ccfDNA yield is dependent on the amount contained in the plasma or serum In general gt 80 of total ccfDNA contained in plasma serum can be obtained using this kit 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 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 Web www epigentek com Printed 2015 06 16 Epigentek Group Inc All rights reserved Products are for research use only P 1064 KIT CONTENTS Component Cat P 1064 2
3. minutes at room temperature to allow DNA to bind to beads Put the tube plate on the EpiMag HT 96 Well Magnetic Separator or an appropriate magnetic stand until the solution is clear about 5 minutes if the magnetic stand is not suitable for the tube plate transfer the beads solution to an appropriate tube or plate well that is compatible to your magnetic stand Carefully remove and discard the supernatant Caution Be careful not to disturb or discard the beads that contain DNA 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 Epigentek Group Inc All rights reserved Products are for research use only Printed 2015 06 16 P 1064 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 Note If EpiMag HT 96 Well Magnetic Separator is used for 0 2 or 0 5 ml tubes the adaptor should be used See the instructions for use of the EpiMag HT 96 Well Magnetic Separator Cat Q10002 1 Add 200 ul of 90 ethanol solution to each tube well and resuspend the beads Place the tube plate on the magnetic stand for 1 minute or until the solution is clear Remove and discard supernatant Add 200 ul of 90 ethanol solution to each tube well Place the tube plate on the magnetic stand for 1 minute or until the
4. 5 Cat P 1064 50 Storage 25 Isolations 50 Isolations Upon Receipt ccfDNA Capture Beads 4ml 8 ml 4 C ccfDNA Capture Enhancer 0 7 ml 1 4 ml 4 C Capture Buffer 23 ml 23 ml X 2 4 C Digestion Solution 2ml 4ml 4 C Proteinase K 60 ul 120 ul 4 C MQ Binding Beads 3 ml 6 ml 4 C Elution Buffer 0 6 ml 1 2 ml 4 C User Guide 1 1 RT Spin the solution down to the bottom prior to use SHIPPING amp STORAGE The kit is shipped in two parts the first part at ambient room temperature and the second part on frozen ice packs at 4 C Each component of the kit is sufficient for the indicated isolation quantity using the standard input amount 0 5 ml per sample Upon receipt Store the following components at 4 C ccfDNA Capture Beads ccfDNA Capture Enhancer Capture Buffer Digestion Solution Proteinase K MQ Binding Beads and Elution Buffer Store all other components at room temperature MATERIALS REQUIRED BUT NOT SUPPLIED Vortex mixer Thermocycler with 48 or 96 well block Pipettes and pipette tips 0 2 ml or 0 5 ml PCR tube 96 well microplate optional 90 Ethanol 0 0 O 0 0 0 OF 0 O Plasma or serum sample 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 Agilent Bioanalyzer or comparable method to assess the size of DNA
5. R E m C D z ped Fig 3 High recovery of ccfDNA confirmed by 2 i bioanalyzer analysis Different amounts of HeLa mononucleosome were spiked into 0 5 ml of plasma then isolated red 200 ng deep blue and a green 500 ng sky blue 500 ng of unpurified HeLa mononucleosome as the control Isolated DNA fragment size 170 bps ow e e E R Ss oo be h A a M Fig 4 High selectivity of specifically isolating small size ccfDNA mononucleosomal DNA Panel A 500 ng of unspiked control polynucleosome up to 2000 bps Panel B 500 ng of the same polynucleosome spiked in 0 5 ml plasma Panel C 500 ng of mononucleosome spiked in 0 5 ml plasma Panel D 500 ng of polynucleosome and 500 ng of mononucleosome which were simultaneously spiked into 0 5 ml plasma ASSAY PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Both fresh and frozen plasma serum from various sources can be used However fresh plasma serum will generally give higher DNA yields than frozen Furthermore frozen plasma serum will lead to DNA loss of about 10 per year The input volume of plasma serum can be from 0 1 1 ml with the standard volume of 0 5 ml per sample If serum sample is used the serum should be prepared within 6 hours after blood draw since lysis of peripheral blood lymphocytes may cause an artificial increase in the amount of DNA during serum separation For the magn
6. UCTS DNA Isolation and Cleanup P 1003 P 1006 P 1007 P 1009 P 1017 P 1018 P 1020 PCR Analysis P 1028 P 1029 DNA Library Prep Magnetic Devices Methylamp MS qPCR Fast Kit EpiQuik Quantitative PCR Fast Kit FitAmp General Tissue Section DNA Isolation Kit DNA Concentrator Kit FitAmp Gel DNA Isolation Kit FitAmp Paraffin Tissue Section DNA Isolation Kit FitAmp Urine DNA Isolation Kit FitAmp Blood and Cultured Cell DNA Extraction Kit FitAmp General DNA Quantification Kit EpiNext DNA Library Preparation Kit Illumina EpiNext High Sensitivity DNA Library Preparation Kit Illumina EpiNext Post Bisulfite DNA Library Preparation Kit Illumina EpiNext High Sensitivity Bisulfite Seq Kit Illumina EpiNext DNA Size Selection Kit EpiNext DNA Purification HT System Q10002 EpiMag HT 96 Well Magnetic Separator 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 2015 06 16 P 1064
7. cell free DNA ccfDNA in plasma serum or other body fluids provides unique opportunities for early detection of a wide range of clinical disorders such as cancer autoimmune disease infection and fetal disorders It was demonstrated that ccfDNA of clinical importance occurs predominantly as fragments of approximately 170 bases from mononucleosomes with a smaller proportion as fragments of 360 bases from di nucleosomes 1 2 Such nucleosomal complexes are released into blood circulation during apoptotic cell death and will be increased under various pathological circumstances such as inflammation pulmonary embolism autoimmune disease and cancer 3 4 It is also shown that using ccfDNA from such nucleosomal complexes for genetic or epigenetic analysis provides better and more accurate identification of physiological and pathological status 5 There are several methods currently being used for ccfDNA isolation from plasma and serum All of these methods are based on capture of DNA by silicone column binding or phenol chloroform separation The DNA isolated by these methods contains both ccfDNA and non ccfDNA which may affect the accuracy of downstream analysis To address these problems Epigentek offers the EpiQuik Circulating Cell Free DNA Isolation Kit for ccfDNA isolation The kit has the following features e Uses innovative magnetic bead based size fractionation technology for selective isolation of circulating cell free DNA from plasma seru
8. etic stand used for capturing DNA bound to magnetic beads we recommend using Epigentek s EpiMag HT 96 Well Magnetic Separator Cat Q10002 1 which has very strong magnetic intensity for 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 2015 06 1 Epigentek Group Inc All rights reserved Products are for research use only rinteg 20 ree AR quickly and efficiently achieving high reproducible retention of magnetic bead bound DNA in various 96 well plates The separator can also be used with 1 7 ml microcentrifuge tubes with volumes greater than 300 ul 1 ccfDNA Capture Add maximum 0 5 ml of plasma serum into each 1 7 ml microcentrifuge tube followed by adding 24 ul of ccfDNA Capture Enhancer 900 ul of Capture Buffer and 50 ul of ccfDNA Capture Beads make sure the beads have been thoroughly resuspended before use Mix by pipetting up and down at least 20 times and incubate at room temperature for 10 min If more than 1 ml of plasma serum from the same sample is used use additional tubes with a maximum of 0 5 ml plasma serum per tube Place the tube in a bench top centrifuge ex Eppendorf 5415D and centrifuge at 12000 rpm for 5 min to spin down the beads to the side of the tube s bottom Place the tip of the tube into the wells of the EpiMag HT 96 Well Magnetic Separator Epigentek Cat Q10002 1 until it is firm
9. ly in place or with an appropriate magnetic separation stand to carefully remove and discard the supernatant Caution Be careful not to disturb or discard the beads that contain DNA While the tube is on the magnetic separator prepare DNA Release Solution by adding 2 ul of Proteinase K to each 40 ul of Digestion Solution Keep the tube in the magnetic stand and add 40 ul of DNA Release Solution to each tube Resuspend the beads This can be done by repeatedly pipetting the 40 ul of DNA Release Solution up and down onto the beads attached to the wall of the tube Transfer the beads solution to a 0 2 ml or 0 5 ml PCR tube Incubate at 55 C for 10 minutes to release the DNA from the beads Capture the beads by placing the tube into the EpiMag HT 96 Well Magnetic Separator or an appropriate magnetic stand until the solution is clear about 5 minutes if the magnetic stand is not suitable for the tube transfer the beads solution to an appropriate tube or plate well that is compatible to your magnetic stand Carefully transfer the supernatant that contains DNA to a new 0 2 ml or 0 5 ml PCR tube or a U bottom 96 well microplate Caution DO NOT discard the supernatant Discard the beads ccfDNA Purification Resuspend MQ Binding Beads by vortexing or shaking Add 2X 2 1 ratio resuspended beads to the DNA sample ex 80 ul of MQ beads to 40 ul of DNA solution Mix thoroughly by pipetting up and down at least 10 times Incubate for 5
10. m that is mainly 170 bps in size The isolated DNA can be directly used for both qPCR and NGS DNA library preparation e Fast and straightforward procedure can be finished within 2 hours No gels columns or centrifugation is needed e Efficient removal of proteins salts nucleases PCR inhibiting substances and other impurities such as polysaccharides polyphenols and lipids 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 2015 06 16 Epigentek Group Inc All rights reserved Products are for research use only P 1064 e Sensitive and efficient DNA capture enables successful isolation with high recovery gt 80 of input monoucleosomal DNA even when the quantities of starting material are limited as low as 0 1 ml e Manual and automation friendly Scalable for single tube or 96 well plate formats References Jahr S et al Cancer Res 2001 61 1659 1665 Suzuki N et al Clin Chim Acta 2008 387 55 58 Holdenrieder S et al Crit Rev Clin Lab Sci 2009 46 1 24 Schwarzenbach H et al Nat Rev Cancer 2011 11 426 437 Chan KCA et al Clinical Chem 2004 50 88 92 oP ON PRINCIPLE amp PROCEDURE The EpiQuik Circulating Cell Free DNA Isolation Kit contains all components which have been optimized for the simple and rapid isolation of small size nucleosomal DNA from plasma serum The mono and di nucleoso
11. mal complexes are efficiently captured via size fractionation magnetic beads ccfDNA Capture Beads by applying the beads to a magnetic field EpiMag HT 96 Well Magnetic Separator Cat Q10002 1 or similar The captured nucleosomal DNA is then enzymatically released and purified using MQ Binding Beads by simply washing the beads The purified ccfDNA is then eluted from the beads for immediate use or storage Plasma serum F Binding of nucleosomal DNA iii D 120 O Control to size fractionation E Spiked in Plasma beads 100 4 N 4 a 80 4 Release of DNA z from the beads 60 4 ral S 8 40 c x 204 Purification of DNA by MQ binding beads 0 T T T 1 4 10 50 200 500 Mononucleosome Amount ng Elution of DNA Fig 2 High recovery of ccfDNA Different amounts of HeLa mononucleosomes were spiked into 0 5 ml of plasma then isolated a using the EpiQuik Circulating Cell Free DNA Isolation Kit The Fig 1 Workflow of the EpiQuik Circulating isolated DNA was fluorescently quantified using control DNA which Cell Free DNA Isolation Kit was directly isolated from the same amount of mononucleosomes 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 2015 06 16 Epigentek Group Inc All rights reserved Products are for research use only P 1064 9 x so xo wo xo ww ko 2 xw w w w w mw E
12. ples for a long time or the sample itself contains a low amount of ccfDNA Increase the volume of the sample for re isolation Improper storage of the kit Ensure that the kit has not exceeded the expiration date The standard shelf life when stored properly is 6 months from date of receipt 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 7 Printed 2015 06 16 P 1064 ccfDNA Capture Beads are not well suspended at step d of ccfDNA capture Completely suspend the beads to allow maximal DNA release Improper ratio of MQ beads to DNA volume during purification Check if the correct volume of MQ Binding Beads was added to the DNA solution at Step 2a Proper ratios should capture fragments gt 100 bps DNA degradation due to improper anticoagulant in blood tube Use new blood sample in EDTA blood tube for plasma serum separation Sample has been subjected to too many freeze thaw cycles Repeated sample freezing and thawing may lead to DNA degradation Always use fresh samples or samples thawed only once Low percentage ethanol used at DNA purification steps Freshly prepared 90 ethanol should be used Presence of larger fragments gt 5 000 bps than expected Lysis of peripheral blood lymphocytes during plasma serum separation The serum should be prepared as soon as possible after blood draw and the separation time short no more than 6 h RELATED PROD
13. solution is clear Remove and discard supernatant Make sure that the ethanol is completely removed after the last wash Air dry beads at room temperature for 2 3 minutes while the tube is on the magnetic stand It is important to ensure all traces of ethanol are removed Note Take care not to over dry the bead spot an over dried bead spot appears cracked as this will significantly decrease elution efficiency Resuspend the beads in 20 ul Elution Buffer and incubate at room temperature for 4 minutes to release the DNA from the beads Capture the beads by placing the tube plate on the magnetic stand for 2 minutes or until the solution is completely clear Note It is normal to see that the eluted solution may be slightly yellow Transfer the supernatant to a new 0 2 ml PCR tube or PCR plate and measure the amount of DNA using a fluorescent method ex use Epigentek s FitAmp General DNA Quantification Kit Cat P 1020 or Picogreen assay If necessary the fragment size of the isolated DNA can be measured using an Agilent Bioanalyzer or comparable method The purified ccfDNA can now be used for a downstream application or stored at 20 C for later use TROUBLESHOOTING Problem Possible Cause Suggestion Low yield of isolated Insufficient amount of starting Increase the volume of plasma serum DNA material for ccfDNA isolation Low concentration of ccfDNA in Sample was left at room temperature the sam
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