Mag-Bind® FFPE DNA Kit Mag-Bind® FFPE DNA - Omega Bio-Tek
Contents
1. 10 minutes to pellet the tissue Note If the tissue does not form a tight pellet centrifuge for an additional 5 minutes 17 10 11 12 13 14 15 18 Mag Bind FFPE DNA Protocols for M6958 Aspirate and discard the xylene Do not disturb the tissue pellet Add 1 mL 100 ethanol Vortex for 20 seconds to mix thoroughly Centrifuge at 4 000 x g for 5 minutes to pellet the tissue The pellet should appear opaque Aspirate and discard the ethanol Do not disturb the tissue pellet Remove any liquid drops with a pipette Repeat Steps 6 8 for a second ethanol wash step Let sit at room temperature for 10 20 minutes Note It is critical to completely dry the sample before the next Proteinase K digestion step Residual ethanol will affect the efficiency of the Proteinase K digestion If a vacuum oven is available place the tube in the vacuum oven preset at 45 C for 10 20 minutes Add 250 uL FTL Buffer and 20 uL Proteinase K Solution Resuspend the pellet by vortexing or pipetting up and down 20 times Incubate at 55 C for 3 5 hours with occasional mixing If necessary extend the incubation to overnight or until the tissue is completely lysed Incubate at 90 C for 45 60 minutes Centrifuge at 4 000 x g for 5 minutes Transfer 200 uL cleared supernatant into a new 1 2 mL or 2 0 mL round well plate 16 17 18 19 20 21 22 23 24 25 Mag Bind FFPE DNA Protocols for M6952. discard the cleared supernatant Do not disturb the Mag Bind Particles CNR Repeat Steps 24 26 for a second DNA Wash step Leave the plate on the magnetic separation device for 5 10 minutes to air dry the Mag Bind Particles CNR Remove any residual liquid with a pipettor Add 30 50 uL Elution Buffer Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 30 times Incubate at room temperature for 10 minutes Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a nuclease free 96 well microplate Store the DNA at 20 C Troubleshooting Guide Please use this guide to troubleshoot any problems that may arise For further assistance please contact the technical support staff toll free at 1 800 832 8896 Low DNA yields Incomplete resuspension of magnetic particles DNA degraded during sample storage MPW Buffer and DNA Wash Buffer were not prepared correctly Loss of magnetic beads during operation Resuspend the magnetic particles by vortexing before use Make sure the sample is properly stored and make sure the samples are processed immediately after collection or removal from storage Prepare MPW Buffer and DNA Wash Buffer according to the instructions on Page 5 Increase the bead collecti
3. uL MPW Wash Buffer Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Note MPW Wash Buffer must be diluted with isopropanol prior to use Please see Page 5 for instructions Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the cleared supernatant Remove any liquid drops from each well Do not disturb the Mag Bind Particles CNR Remove the tube containing the Mag Bind Particles CNR from the magnetic separation device 25 26 27 28 29 30 31 32 33 Mag Bind FFPE DNA Protocols for M6957 Add 400 uL DNA Wash Buffer Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Note DNA Wash Buffer must be diluted with ethanol prior to use Please see Page 5 for instructions Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CNR Repeat Steps 24 26 for a second DNA Wash step Leave the tube on the magnetic separation device for 5 10 minutes to air dry the Mag Bind Particles CNR Remove any residual liquid with a pipettor Remove the tube containing the Mag Bind Particles CNR from the magnetic separation device Add 30 50 uL Elution Buffer Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 30 times Incub
4. 8 Add 500 uL MB3 Buffer and 10 uL Mag Bind Particles CNR Mix thoroughly by vortexing or pipetting up and down 10 20 times Note If DNA content from sample is expected to be low add 10 uL LPA Let sit at room temperature for 5 10 minutes Place the plate on the magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Remove the plate from the magnetic separation device Add 400 uL MPW Wash Buffer Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Note MPW Wash Buffer must be diluted with isopropanol prior to use Please see Page 5 for instructions Place the plate on the magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Remove the plate from the magnetic separation device Add 400 uL DNA Wash Buffer Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Note DNA Wash Buffer must be diluted with ethanol prior to use Please see Page 5 for instructions 19 26 27 28 29 30 31 32 20 Mag Bind FFPE DNA Protocols for M6958 Aspirate and
5. Bind Particles CNR by vortexing or pipetting up and down 20 times Note DNA Wash Buffer must be diluted with ethanol prior to use Please see Page 5 for instructions Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CNR Repeat Steps 20 22 for a second DNA Wash step Leave the tube on the magnetic separation device for 5 10 minutes to air dry the Mag Bind Particles CNR Remove any residual liquid with a pipettor Remove the tube containing the Mag Bind Particles CNR from the magnetic separation device Add 30 50 uL Elution Buffer Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 30 times 27 28 29 Mag Bind FFPE DNA Protocols for M6957 Incubate at room temperature for 10 minutes Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a clean 1 5 mL microcentrifuge tube Store the DNA at 20 C Mag Bind FFPE DNA Protocols for M6957 Mag Bind FFPE DNA 1 5 mL microcentrifuge tube with xylene Note The following protocol uses xylene to remove paraffin from the FFPE sample Use fume hood and take proper protection during xylene extraction Materials and Equipment to be Supplied by User Microcentrifuge capable of gt 14 000 x g Magnetic separ
6. Ca OMEGA Innovations in nucleic acid isolation bio tek Product Manual Mag Bind FFPE DNA Kit M6957 00 5 preps M6957 01 50 Preps M6957 02 200 Preps Mag Bind FFPE DNA 96 Kit M6958 00 1 x 96 preps M6958 01 4 x 96 preps July 2013 For research use only Not intended for diagnostic testing Mag Bind FFPE DNA Kit Mag Bind FFPE DNA 96 Kit Table of Contents Introduction and OVEFVIEW sscsssccsscsecsseessecseccnsecseecnsceneerses 2 Kit Content ae 3 Important Note uniones 4 Prepaid Reagan een 5 Mag Bind FFPE DNA Protocol ussessesossonesnssenssensesnsennssensene 6 Mag Bind FFPE DNA Protocol with Xylene 10 Mag Bind FFPE DNA 96 Protocol 14 Mag Bind FFPE DNA 96 Protocol with Xylene 17 Troubleshooting Guide nennen 21 Orla 22 Manual Revision July 2013 Fi OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview Introduction The Mag Bind FFPE DNA and the Mag Bind FFPE DNA 96 kits provide a rapid and easy method for the isolation of total DNA from formalin fixed paraffin embedded FFPE tissue sections Due to fixation and embedding procedures nucleic acids in FFPE samples are heavily fragmented and modified by formaldehyde While the Mag Bind FFPE DNA and the Mag Bind FFPE DNA 96 kits are optimized to minimize the effect of the formaldehyde modification it is not recommended to use the DNA purified with these kits for downstream a
7. NR are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a nuclease free 96 well microplate Store the DNA at 20 C Mag Bind FFPE DNA Protocols for M6958 Mag Bind FFPE DNA 96 96 well plate with xylene Note The following protocol uses xylene to remove paraffin from the FFPE sample Use fume hood and take proper protection during xylene extraction Materials and Equipment to be Supplied by User Centrifuge with swing bucket rotor capable of 4 000 x g Rotor adaptor for 96 well deep well plates Magnetic separation device for 96 well deep well plates Cat MSD 01B Water bath or heat block capable of 55 C Water bath or heat block capable of 80 C Water bath or heat block capable of 90 C Vortexer 1 2 mL or 2 0 mL round well plates Nuclease free 96 well microplates e Isopropanol 100 Ethanol e Xylene Sealing film Things to do before starting Prepare Buffers according to Preparing Reagents section on Page 5 Set water baths or heat blocks to 90 C 80 C and 55 C Vortex the Mag Bind Particles CNR thoroughly before use 1 Add 1 mL xylene into each well of a 1 2 mL or 2 0 mL round well plate 2 Cut 3 8 paraffin sample sections between 5 10 um Note Do not use the first 2 3 sections from the sample block 3 Immediately add 2 5 sections to the xylene Vortex for 20 seconds to mix thoroughly 4 Centrifuge at 4 000 x g for 5
8. at 2 8 C Proteinase K Solution can be stored at room temperature for 12 months For long term storage gt 12 months store at 2 8 C Store all other components at room temperature 22 25 C Check buffers for precipitates before use Redissolve any precipitates by warming to 37 C Starting Materials Since standard formalin fixation and paraffin embedding procedures cause significant fragmentation of nucleic acids we recommend following guidelines to limit the extent of DNA RNA fragmentation 1 Use 4 10 formalin to fixate tissue samples 2 Limit the fixation time to 14 24 hours 3 Completely dehydrate samples before embedding Always use freshly cut sections of FFPE tissue for DNA isolation For the first time user we recommend to use less than 3 5 sections with thickness of 10 um Depending on the yield and purity obtained it may be possible to increase the starting material Preparing Reagents Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature Kit 100 Ethanol to be Added Dilute MPW Buffer with isopropanol as follows and store at room temperature Kit Isopropanol to be Added M6958 01 125 mL Mag Bind FFPE DNA Protocols for M6957 Mag Bind FFPE DNA Protocol 1 5 mL microcentrifuge tube Materials and Equipment to be Supplied by User Microcentrifuge capable of gt 14 000 x g Magnetic separation device for 1 5 mL microcentrifuge tubes Cat MSD 02 Water bath or heat blo
9. ate at room temperature for 10 minutes Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a clean 1 5 mL microcentrifuge tube Store the DNA at 20 C 13 Mag Bind FFPE DNA Protocols for M6958 Mag Bind FFPE DNA 96 96 well plate Materials and Equipment to be Supplied by User Centrifuge with swing bucket rotor capable of 4 000 x g Rotor adaptor for 96 well deep well plates Magnetic separation device for 96 well deep well plates Cat MSD 01B Water bath or heat block capable of 55 C Water bath or heat block capable of 80 C Water bath or heat block capable of 90 C Vortexer 1 2 mL or 2 0 mL round well plates Nuclease free 96 well microplates Isopropanol 100 Ethanol Sealing film Things to do before starting 14 Prepare Buffers according to Preparing Reagents section on Page 5 Set water baths or heat blocks to 90 C 80 C and 55 C Vortex the Mag Bind Particles CNR thoroughly before use Add 250 uL FTL Buffer into each well of a 1 2 mL or 2 0 mL round well plate Cut 3 8 paraffin sample sections between 5 10 um Note Do not use the first 2 3 sections from the sample block Immediately add 2 5 sections to the FTL Buffer Centrifuge at 4 000 x g for 5 minutes at room temperature Incubate at 80 C fo
10. ation device for 1 5 mL microcentrifuge tubes Cat MSD 02 Water bath or heat block capable of 55 C Water bath or heat block capable of 80 C Water bath or heat block capable of 90 C Vortexer Nuclease free 1 5 mL microcentrifuge tubes Isopropanol 100 Ethanol Xylene Things to do before starting 10 Prepare Buffers according to Preparing Reagents section on Page 5 Set water baths or heat blocks to 90 C 80 C and 55 C Vortex the Mag Bind Particles CNR thoroughly before use Add 1 mL xylene into a new 1 5 mL microcentrifuge tube Cut 3 8 paraffin sample sections between 5 10 um Note Do not use the first 2 3 sections from the sample block Immediately add 2 5 sections to the xylene Vortex for 20 seconds to mix thoroughly Centrifuge at maximum speed 214 000 x g for 5 10 minutes to pellet the tissue Note If the tissue does not form a tight pellet centrifuge for an additional 3 minutes 10 11 12 13 14 15 Mag Bind FFPE DNA Protocols for M6957 Aspirate and discard the xylene Do not disturb the tissue pellet Add 1 mL 100 ethanol Vortex for 20 seconds to mix thoroughly Centrifuge at maximum speed 214 000 x g for 5 minutes to pellet the tissue The pellet should appear opaque Aspirate and discard the ethanol Do not disturb the tissue pellet Remove any liquid drops with a pipette Repeat Steps 6 8 for a second ethanol wash step Let sit at room temperature for 10 20 minu
11. ck capable of 55 C Water bath or heat block capable of 80 C Water bath or heat block capable of 90 C Vortexer Nuclease free 1 5 mL microcentrifuge tubes Isopropanol 100 Ethanol Before Starting Prepare Buffers according to Preparing Reagents section on Page 5 Set water baths or heat blocks to 90 C 80 C and 55 C Vortex the Mag Bind Particles CNR thoroughly before use 1 Add 250 uL FTL Buffer into a new 1 5 mL microcentrifuge tube 2 Cut 3 8 paraffin sample sections between 5 10 um Note Do not use the first 2 3 sections from the sample block 3 Immediately add 2 5 sections to the FTL Buffer 4 Centrifuge at maximum speed 214 000 x g for 60 seconds 5 Incubate at 80 C for 15 minutes Mix the sample a few times by gently shaking the tube 2 3 times Make sure that the tissue sections stay submerged in the solution 6 Let sit at room temperature for 5 minutes 10 11 12 13 14 13 16 Mag Bind FFPE DNA Protocols for M6957 Add 20 uL Proteinase K Solution Incubate at 55 C for 3 5 hours with occasional mixing If necessary extend the incubation to overnight or until the tissue is completely lysed Incubate at 90 C for 45 60 minutes Centrifuge at maximum speed 214 000 x g for 5 minutes The paraffin will form a thin layer on top of the lysate solution Transfer 200 uL cleared lysate into a new 1 5 mL microcentrifuge tube Tip Use a 1 mL pipette tip o
12. g Bind Particles CNR by vortexing or pipetting up and down 20 times Note MPW Wash Buffer must be diluted with isopropanol prior to use Please see Page 5 for instructions 15 18 19 20 21 22 23 24 25 26 27 28 16 Mag Bind FFPE DNA Protocols for M6958 Place the plate on the magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Remove the plate from the magnetic separation device Add 400 uL DNA Wash Buffer Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Note DNA Wash Buffer must be diluted with ethanol prior to use Please see Page 5 for instructions Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CNR Repeat Steps 20 22 for a second DNA Wash step Leave the plate on the magnetic separation device for 5 10 minutes to air dry the Mag Bind Particles CNR Remove any residual liquid with a pipettor Add 30 50 uL Elution Buffer Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 30 times Incubate at room temperature for 10 minutes Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles C
13. on time Problem with downstream application Carryover of the magnetic beads in the elution DNA is over fixated during tissue formalin fixation Carryover the magnetic beads in the eluted DNA will not effect downstream applications Extend incubation time at 90 C to 90 minutes To remove the carryover magnetic particles from the eluted DNA simply magnetize the magnetic particles and carefully transfer the DNA eluate to a new plate 21 22 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 a A 96 well Microplate 300 pL 5 pk 96 well Microplate 300 uL 25 pk 96 well Microplate 500 uL 25 pk HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc Qiagen QlAvac and Vacman are all trademarks of their respective companies PCRis a patented process of Hoffman La Roche Use of the PCR process requires a license Notes 23 Notes 24
14. pplications that require full length DNA Overview The Mag Bind FFPE DNA and the Mag Bind FFPE DNA 96 kits combine the high efficiency binding properties of Mag Bind technology with a specially designed buffer system to isolate total DNA sample from FFPE samples There are two protocols included in this manual The standard protocol uses a heating step to remove paraffin from the sample The alternative protocol uses the traditional xylene extraction to remove paraffin After the paraffin removal steps samples are first lysed in FTL Buffer with digestion of Proteinase K The lysate is then heated to denature the proteinase and mixed with MB3 Buffer and magnetic particles to bind the nucleic acid on the surface of the Mag Bind particles After two wash steps purified DNA is eluted with Elution Buffer or nuclease free water New in this Edition This manual has been edited for content and redesigned to enhance user readability Proteinase K is now supplied in a liquid form eliminating the resuspension step to prior to use Proteinase K Solution can also be stored at room temperature for 12 months Proteinase Storage Buffer is no longer included in the kit Kit Contents 7 Important Notes Storage and Stability All the Mag Bind FFPE DNA Kit and the Mag Bind FFPE DNA 96 Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows Mag Bind Particles CNR should be stored
15. r 15 minutes Mix the sample a few times by gently shaking the plate 2 3 times Make sure that the tissue sections stay submerged in the solution Note Seal the plate with sealing film to prevent evaporation during incubation 10 11 12 13 14 15 16 17 Mag Bind FFPE DNA Protocols for M6958 Let sit at room temperature for 5 minutes Add 20 uL Proteinase K Solution Incubate at 55 C for 3 5 hours with occasional mixing If necessary extend the incubation to overnight or until the tissue is completely lysed Incubate at 90 C for 45 60 minutes Centrifuge at 4 000 x g for 5 minutes The paraffin will form a thin layer on top of the lysate solution Transfer 200 uL cleared lysate into a new 96 well round well plate Tip Use a 1 mL pipette tip or large orifice tip to penetrate the paraffin layer Add 500 uL MB3 Buffer and 10 uL Mag Bind Particles CNR Mix thoroughly by vortexing or pipetting up and down 10 20 times Note If DNA content from sample is expected to be low add 10 uL LPA Let sit at room temperature for 5 10 minutes Place the plate on the magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Remove the plate from the magnetic separation device Add 400 uL MPW Wash Buffer Resuspend the Ma
16. r large orifice tip to penetrate the paraffin layer Add 500 uL MB3 and 10 uL Mag Bind Particles CNR Mix thoroughly by vortexing or pipetting up and down 10 20 times Note If DNA content from sample is expected to be low add 10 uL LPA Let sit at room temperature for 5 10 minutes Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CNR Remove the tube containing the Mag Bind Particles CNR from the magnetic separation device 17 18 19 20 21 22 23 24 25 26 Mag Bind FFPE DNA Protocols for M6957 Add 400 uL MPW Wash Buffer Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Note MPW Wash Buffer must be diluted with isopropanol prior to use Please see Page 5 for instructions Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the cleared supernatant Remove any liquid drops from each well Do not disturb the Mag Bind Particles CNR Remove the tube containing the Mag Bind Particles CNR from the magnetic separation device Add 400 uL DNA Wash Buffer Resuspend the Mag
17. tes Note It is critical to completely dry the sample before the next Proteinase K digestion step Residual ethanol will affect the efficiency of the Proteinase K digestion If a vacuum oven is available place the tube in the vacuum oven preset at 45 C for 10 20 minutes Add 250 uL FTL Buffer and 20 uL Proteinase K Solution Resuspend the pellet by vortexing or pipetting up and down 20 times Incubate at 55 C for 3 5 hours with occasional mixing If necessary extend the incubation to overnight or until the tissue is completely lysed Incubate at 90 C for 45 60 minutes Centrifuge at maximum speed 214 000 x g for 5 minutes Transfer 200 uL cleared supernatant into a new 1 5 mL microcentrifuge tube 11 16 17 18 19 20 21 22 23 24 12 Mag Bind FFPE DNA Protocols for M6957 Add 500 uL MB3 Buffer and 10 uL Mag Bind Particles CNR Mix thoroughly by vortexing or pipetting up and down 10 20 times Note If DNA content from sample is expected to be low add 10 uL LPA Let sit at room temperature for 5 10 minutes Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CNR Remove the tube containing the Mag Bind Particles CNR from the magnetic separation device Add 400
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