Mag-Bind® Tissue DNA Kit Mag-Bind® Tissue - Omega Bio-Tek
Contents
1. 22 23 24 25 26 Mag Bind Tissue DNA Protocols Let sit for 3 minutes at room temperature Vortex or pipet up and down a few times during incubation Place the tube on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Remove the tube from the magnetic separation device Add 400 uL SPM Wash Buffer Note SPM Wash Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles SC by vortexing or pipetting up and down 20 30 times Let sit for 3 minutes at room temperature Vortex or pipet up and down a few times during incubation Place the tube on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Repeat Steps 18 23 for a second SPM Wash Buffer wash step Leave the tube on the magnetic separation device for 10 minutes to air dry the Mag Bind Particles SC Remove any residual liquid with a pipettor Remove the tube from the magnetic separation device 11 27 28 29 30 31 32 12 Mag Bind Tissue DNA Protocols Add 52. Leave the plate on the magnetic separation device for 10 minutes to air dry the Mag Bind Particles SC Remove any residual liquid with a pipettor Remove the plate from the magnetic separation device Add 50 200 uL Elution Buffer preheated to 70 C Resuspend the Mag Bind Particles SC by vortexing for 3 minutes or pipetting up and down 50 times Let sit at room temperature for 5 10 minutes Place the plate on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a 96 well microplate Store DNA at 20 C Mag Bind Tissue DNA 96 Protocols Mag Bind Tissue DNA 96 Kit Protocol Cultured Cells This protocol is designed for rapid isolation of up to 25 ug genomic DNA from up to 5 x 10 cultured cells Materials and Equipment to be Supplied by User Magnetic separation device for 96 well plates Cat MSD 01B or MSD 01 e 96 well deep well plates compatible with the magnetic separation device 96 well microplates e Centrifuge with swing bucket rotor capable of 4000 x g Centrifuge adaptor for 96 well plates Water bath incubator or heat block capable of 70 C Shaking water bath capable of 55 C Vortexer 100 ethanol Sealing film Multi channel reservoir e Cold PBS 4 C Before Starting Prepare all Reagents ac
3. Bind Particles SC and 10 uL LPA Buffer Vortex or pipet up and down 20 30 times to mix thoroughly Let sit at room temperature for 5 minutes Place the tube on a magnetic separation device to magnetize the Mag Bind Particles SC Let sit for 10 15 minutes Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Remove the tube from the magnetic separation device Add 400 uL MP Buffer Note MP Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions 15 16 17 18 19 20 21 22 23 24 25 Mag Bind Tissue DNA Protocols Resuspend the Mag Bind Particles SC by vortexing or pipetting up and down 20 30 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles SC Let sit for 3 minutes at room temperature Vortex or pipet up and down a few times during incubation Place the tube on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Remove the tube from the magnetic separation device Add 400 uL SPM Wash Buffer Note SPM Wash Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles SC by vortexing or pipetting up and down
4. Particles SC and 10 uL LPA Buffer Mix thoroughly by vortexing or pipetting Let sit at room temperature for 5 minutes Place the plate on a magnetic separation device to magnetize the Mag Bind Particles SC Let sit for 10 15 minutes Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Remove the plate from the magnetic separation device Add 400 uL MP Buffer Note MP Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions 14 15 16 17 18 19 20 21 22 23 Mag Bind Tissue DNA 96 Protocols Resuspend the Mag Bind Particles SC by vortexing or pipetting up and down 20 30 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles SC Let sit for 3 minutes at room temperature Vortex or pipet up and down a few times during incubation Place the plate on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Remove the plate from the magnetic separation device Add 400 uL SPM Wash Buffer Note SPM Wash Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles SC by vortexing or pipetting up and down 20 30 times Note Complete
5. Particles SC are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Repeat Steps 20 25 for a second SPM Wash Buffer wash step Leave the plate on the magnetic separation device for 10 minutes to air dry the Mag Bind Particles SC Remove any residual liquid with a pipettor Remove the plate from the magnetic separation device Add 50 200 uL Elution Buffer preheated to 70 C Resuspend the Mag Bind Particles SC by vortexing for 3 minutes or pipetting up and down 50 times Let sit at room temperature for 5 10 minutes Place the plate on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a 96 well microplate Store DNA at 20 C Mag Bind Tissue DNA 96 Protocols Mag Bind Tissue DNA 96 Kit Protocol Buccal Swabs Materials and Equipment to be Supplied by User Magnetic separation device for 96 well plates Cat MSD 01B or MSD 01 96 well deep well plates compatible with the magnetic separation device e 96 well microplates e Centrifuge with swing bucket rotor capable of 4000 x g e Centrifuge adaptor for 96 well plates Water bath incubator or heat block capable of 70 C Shaking water bath capable of 55 C Vortexer 100 ethanol e Sealing film e Multi
6. Transfer the cleared lysate to a new plate OPTIONAL Certain tissues such as liver have high levels of RNA which will be purified with DNA using this kit While it will not interfere with PCR the RNA can be removed at this point Add 5 uL RNase A assuming a sample size of 10 mg and let sit at room temperature for 2 minutes Proceed to Step 5 5 Centrifuge at maximum speed for 5 minutes to pellet undigested tissue debris and hair 6 Carefully transfer 200 uL of the supernatant to a new 96 well deep well plate without disturbing the undigested pellet 7 Add 200 uL MSL Buffer Vortex or pipet up and down to mix thoroughly 8 Incubate at 70 C for 10 minutes 9 Place the plate on the bench for 5 minutes to bring the plate to room temperature 10 Add 290 uL 100 ethanol and 10 uL Mag Bind Particles SC Vortex or pipet up and down 20 30 times to mix thoroughly 11 Let sit at room temperature for 5 minutes 12 Place the plate on a magnetic separation device to magnetize the Mag Bind Particles SC Let sit for 10 15 minutes 22 13 14 15 16 17 18 19 20 21 22 Mag Bind Tissue DNA 96 Protocols Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Remove the plate from the magnetic separation device Add 400 uL MP Buffer Note MP Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles SC by
7. Wash Buffer by adding not prepared correctly ethanol according to the instructions Loss of magnetic beads Be careful not to disturb the Mag Bind during operation Particles SC during aspiration steps Inefficient cell lysis due to decrease of activity Add more Proteinase K Solution of Proteinase K SPM Wash Buffer was not diluted with 100 ethanol Use more stating material Quantify the purified DNA accurately and use sufficient DNA Excess DNA was used Make sure to use correct amount DNA Resuspend the Mag Bind Particles SC by vortexing before use Prepare SPM Wash Buffer as instructed on the Page 4 Insufficient DNA was used 37 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 C HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license 38
8. discard the supernatant Do not disturb the Mag Bind Particles SC Repeat Steps 20 25 for a second SPM Wash Buffer wash step Leave the plate on the magnetic separation device for 10 minutes to air dry the Mag Bind Particles SC Remove any residual liquid with a pipettor Remove the plate from the magnetic separation device 35 29 30 31 32 33 34 36 Mag Bind Tissue DNA 96 Protocols Add 50 200 uL Elution Buffer preheated to 70 C Resuspend the Mag Bind Particles SC by vortexing for 3 minutes or pipetting up and down 50 times Let sit at room temperature for 5 10 minutes Place the plate on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a 96 well microplate Store 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 Problem Low DNA yields Problem No DNA eluted Problem Problem with downstream applications Incomplete resuspension of Mag Bind Particles SC Inefficient cell lysis due i y Make sure the sample is thoroughly mixed to inefficient mix of MSL Buffer and sample Witi MSE Buffer SPM Wash Buffer were Prepare the SPM
9. resuspension is required for adequate washing of the Mag Bind Particles SC Let sit for 3 minutes at room temperature Vortex or pipet up and down a few times during incubation Place the plate on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC 27 24 25 26 27 28 29 30 31 32 28 Mag Bind Tissue DNA 96 Protocols Repeat Steps 18 23 for a second SPM Wash Buffer wash step Leave the plate on the magnetic separation device for 10 minutes to air dry the Mag Bind Particles SC Remove any residual liquid with a pipettor Remove the plate from the magnetic separation device Add 50 200 uL Elution Buffer preheated to 70 C Resuspend the Mag Bind Particles SC by vortexing for 3 minutes or pipetting up and down 50 times Let sit at room temperature for 5 10 minutes Place the plate on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a 96 well microplate Store DNA at 20 C Mag Bind Tissue DNA 96 Protocols Mag Bind Tissue DNA 96 Kit Protocol Mouse Tail Snips Materials and Equipment to be Su
10. vortexing or pipetting up and down 20 30 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles SC Let sit for 3 minutes at room temperature Vortex or pipet up and down a few times during incubation Place the plate on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Remove the plate from the magnetic separation device Add 400 uL SPM Wash Buffer Note SPM Wash Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles SC by vortexing or pipetting up and down 20 30 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles SC 23 23 24 25 26 27 28 29 30 31 32 33 34 24 Mag Bind Tissue DNA 96 Protocols Let sit for 3 minutes at room temperature Vortex or pipet up and down a few times during incubation Place the plate on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Repeat Steps 18 23 for a second SPM Wash Buffer wash step
11. x g Centrifuge adaptor for 96 well plates Water bath incubator or heat block capable of 70 C Shaking water bath capable of 55 C Vortexer 100 ethanol Sealing film Multi channel reservoir Before Starting Prepare all Reagents according to Preparing Reagents section on Page 4 Preheat Elution Buffer to 70 C Set water bath to 55 C OPTIONAL Although mechanical homogenization of tissue is not necessary pulverizing the samples in liquid nitrogen will improve lysis and reduce incubation time Once the liquid nitrogen has evaporated transfer the powdered tissue to a clean 96 well deep well plate Add 200 uL TL Buffer and proceed to Step 3 below 1 Mince up to 10 mg tissue and transfer to a 96 well deep well plate Note Cutting the tissue into small pieces can speed up lysis 2 Add 250 uLTL Buffer 3 Add 20 uL Proteinase K Solution Vortex to mix thoroughly Note Seal the plate before vortexing to prevent cross contamination or loss of sample 21 Mag Bind Tissue DNA 96 Protocols 4 Incubate at 55 C in a shaking water bath Note If a shaking water bath is not available vortex the sample every 20 30 minutes Lysis time depends on amount and type of tissue but is usually under 3 hours The lysis can proceed overnight Important Some tissue may contain material that can not be digested with proteinase centrifuge the plate at maximum speed for 5 minutes to remove the undigested material
12. 0 200 uL Elution Buffer preheated to 70 C Resuspend the Mag Bind Particles SC by vortexing for 3 minutes or pipetting up and down 50 times Let sit at room temperature for 5 10 minutes Place the tube on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a nuclease free 1 5 mL microcentrifuge tube Store DNA at 20 C Mag Bind Tissue DNA Protocols Mag Bind Tissue DNA Kit Protocol Mouse Tail Snips Materials and Equipment to be Supplied by User Magnetic separation device for 1 5 mL microcentrifuge tubes Cat MSD 02 e Tabletop microcentrifuge e Nuclease free 1 5 mL microcentrifuge tubes Water bath incubator or heat block capable of 70 C Shaking water bath capable of 55 C Vortexer 100 ethanol Before Starting Prepare all Reagents according to Preparing Reagents section on Page 4 Preheat Elution Buffer to 70 C Set shaking water bath to 55 C 1 Snip a 2 5 mm piece of mouse tail cut into several pieces and place the pieces into a nuclease free 1 5 mL microcentrifuge tube Note Follow all regulations regarding the safe and humane treatment of animals Mice should not be older than 6 weeks as lysis will be more difficult in older animals resulting in suboptimal DNA yields If possible obtain tail biopsie
13. 20 30 times Let sit for 3 minutes at room temperature Vortex or pipet up and down a few times during incubation Place the tube on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Repeat Steps 19 24 for a second SPM Wash Buffer wash step 15 26 27 28 29 30 31 32 33 16 Mag Bind Tissue DNA Protocols Leave the tube on the magnetic separation device for 10 minutes to air dry the Mag Bind Particles SC Remove any residual liquid with a pipettor Remove the tube from the magnetic separation device Add 50 200 uL Elution Buffer preheated to 70 C Resuspend the Mag Bind Particles SC by vortexing for 3 minutes or pipetting up and down 50 times Let sit at room temperature for 5 10 minutes Place the tube on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a nuclease free 1 5 mL microcentrifuge tube Store DNA at 20 C Mag Bind Tissue DNA Protocols Mag Bind Tissue DNA Kit Protocol Buccal Swabs Materials and Equipment to be Supplied by User Magnetic separation device for 1 5 mL microcentr
14. Buffer Note MP Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles SC by vortexing or pipetting up and down 20 30 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles SC Let sit for 3 minutes at room temperature Vortex or pipet up and down a few times during incubation 18 19 20 21 22 23 24 25 26 27 28 Mag Bind Tissue DNA 96 Protocols Place the plate on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Remove the plate from the magnetic separation device Add 400 uL SPM Wash Buffer Note SPM Wash Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles SC by vortexing or pipetting up and down 20 30 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles SC Let sit for 3 minutes at room temperature Vortex or pipet up and down a few times during incubation Place the plate on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Aspirate and
15. Ca OMEGA Innovations in nucleic acid isolation bio tek Product Manual Mag Bind Tissue DNA Kit M6223 00 5 preps M6223 02 200 preps Mag Bind Tissue DNA 96 Kit M6229 00 1 x 96 preps M6229 01 4x 96 preps June 2013 For research use only Not intended for diagnostic testing Mag Bind Tissue DNA Kit Mag Bind Tissue DNA 96 Kit Table of Contents Introduction and OVEFVIEW csssssccsscsecssecssecseccssesseecseceseersees 2 Kit Contents Storage and Stability esseessseseseeecnseeesees 3 Preparing Reagents ssessssssssssseeesssseseesssssreeesssseesossssssoosssseeess 4 Mag Bind Tissue DNA Tissue ProtoCoOl ssessesseeserseessesessse 5 Mag Bind Tissue DNA Cultured Cells Protocol 9 Mag Bind Tissue DNA Mouse Tail Snips Protocol 13 Mag Bind Tissue DNA Buccal Swabs Protocol 17 Mag Bind Tissue DNA 96 Tissue ProtoCol ccssecseeees 21 Mag Bind Tissue DNA 96 Cultured Cells Protocol 25 Mag Bind Tissue DNA 96 Mouse Tail Snips Protocol 29 Mag Bind Tissue DNA 96 Buccal Swabs Protocol 33 Troubleshooting Guide sessssssseeesessssssssseeesersssssssseeeseessesssss 37 Iko Inla TO EIA A 38 Manual Revision June 2013 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview The Mag Bind Tissue DNA Kit and the Mag Bind Tissue DNA 96 Kit are designed for rapid and reliable isolation of high qualit
16. L microcentrifuge tubes Cat MSD 02 e Tabletop microcentrifuge Nuclease free 1 5 mL microcentrifuge tubes Water bath incubator or heat block capable of 70 C e Shaking water bath capable of 55 C Vortexer 100 ethanol Before Starting Prepare all Reagents according to Preparing Reagents section on Page 4 Preheat Elution Buffer to 70 C e Set shaking water bath to 55 C OPTIONAL Although mechanical homogenization of tissue is not necessary pulverizing the samples in liquid nitrogen will improve lysis and reduce incubation time Once the liquid nitrogen has evaporated transfer the powdered tissue to a clean 1 5 mL microcentrifuge tube Add 200 uL TL Buffer and proceed to Step 2 below 1 Mince up to 20 mg tissue and place into a 1 5 mL microcentrifuge tube Add 250 uL TL Buffer Note Cutting the tissue into small pieces can speed up lysis 2 Add 20 uL Proteinase K Solution Vortex to mix thoroughly 3 Incubate at 55 C in a shaking water bath Lysis time will depend on the amount and type of tissue but is usually under 3 hours Lysis can proceed overnight Note If a shaking water bath is not available vortex the sample every 20 30 minutes 10 11 12 13 14 Mag Bind Tissue DNA Protocols Centrifuge at 13 000 x g for 5 minutes Carefully transfer 200 uL of the cleared supernatant to a new 1 5 mL microcentrifuge tube Add 200 uL MSL Buffer and 5 uL RNase A Vortex or pipet up
17. and down to mix thoroughly Incubate at 70 C for 10 minutes Place the sample on the bench for 5 minutes to bring the sample to room temperature Add 290 uL 100 ethanol and 10 uL Mag Bind Particles SC Vortex or pipet up and down 20 30 times to mix thoroughly Let sit at room temperature for 5 minutes Optional For samples with an expected yield less than 8 ug add 10 uL LPA Buffer to help increase the yield Place the tube on a magnetic separation device to magnetize the Mag Bind Particles SC Let sit for 10 15 minutes Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Remove the tube from the magnetic separation device Add 400 uL MP Buffer Note MP Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions 15 16 17 18 19 20 21 22 23 24 25 Mag Bind Tissue DNA Protocols Resuspend the Mag Bind Particles SC by vortexing or pipetting up and down 20 30 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles SC Let sit for 3 minutes at room temperature Vortex or pipet up and down a few times during incubation Place the tube on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Par
18. atment or cell scraper Wash cells twice with cold PBS 4 C and resuspend the cells in 180 uL cold PBS Proceed with Step 2 of this protocol 2 Add 20 uL Proteinase K Solution Vortex to mix thoroughly 10 11 12 13 14 10 Mag Bind Tissue DNA Protocols Incubate at 55 C for 10 minutes in a shaking water bath Note If a shaking water bath is not available vortex the sample every 2 3 minutes Transfer the sample to a new 1 5 mL microcentrifuge tube Add 200 uL MSL Buffer Vortex or pipet up and down to mix thoroughly Incubate at 70 C for 10 minutes Place the sample on the bench for 5 minutes to bring the sample to room temperature Add 260 uL 100 ethanol 10 uL Mag Bind Particles SC and 10 uL LPA Buffer Vortex or pipet up and down 20 30 times to mix thoroughly Let sit at room temperature for 5 minutes Place the tube on a magnetic separation device to magnetize the Mag Bind Particles SC Let sit for 10 15 minutes Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Remove the tube from the magnetic separation device Add 400 uL MP Buffer Note MP Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles SC by vortexing or pipetting up and down 20 30 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles SC 15 16 17 18 19 20 21
19. channel reservoir Before Starting e Prepare all Reagents according to Preparing Reagents section on Page 4 Preheat Elution Buffer to 70 C Set shaking water bath to 55 C 1 Cut off the buccal brush or swab head and place each swab into a well of a 96 well deep well plate 2 Add 400 uLTL Buffer 3 Add 20 uL Proteinase K Solution Vortex to mix thoroughly Note Seal the plate before vortexing to prevent cross contamination or loss of sample 4 Incubate at 55 C in a shaking water bath for 45 minutes Note If a shaking water bath is not available vortex the plate every 5 10 minutes 5 Centrifuge at 3000 x g for 10 minutes 33 10 11 12 13 14 15 16 17 34 Mag Bind Tissue DNA 96 Protocols Transfer 200 uL lysate into a new 96 well deep well plate Do not transfer the swabs to the new plate Add 200 uL MSL Buffer Vortex to mix thoroughly Incubate at 70 C for 10 minutes Place the plate on the bench for 5 minutes to bring to room temperature Add 260 uL of 100 ethanol 10 uL Mag Bind Particles SC and 10 uL LPA Buffer Mix thoroughly by vortexing or pipetting Let sit at room temperature for 5 minutes Place the plate on a magnetic separation device to magnetize the Mag Bind Particles SC Let sit for 10 15 minutes Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Remove the plate from the magnetic separation device Add 400 uL MP
20. cording to Preparing Reagents section on Page 4 Preheat Elution Buffer to 70 C Set shaking water bath to 55 C 1 Prepare the cell suspension la Frozen cell samples should be thawed before starting this protocol Pellet cells by centrifugation Wash the cells with cold PBS 4 C and resuspend cells in 180 uL cold PBS Proceed with Step 2 of this protocol 1b For cells grown in suspension pellet 5 x 10 cells at 1 200 x g in a centrifuge tube Discard the supernatant wash the cells once with cold PBS 4 C and resuspend cells in 180 uL cold PBS Proceed with Step 2 of this protocol 1c For cells grown in a monolayer harvest the cells by either using a trypsin treatment or cell scraper Wash cells twice in cold PBS 4 C and resuspend the cells with 180 uL cold PBS Proceed with Step 2 of this protocol 25 10 11 12 13 26 Mag Bind Tissue DNA 96 Protocols Add 20 uL Proteinase K Solution Vortex to mix thoroughly Note Seal the plate before vortexing to prevent cross contamination or loss of sample Incubate at 55 C in a shaking water bath for 10 minutes Note If a shaking water bath is not available vortex the sample every 2 3 minutes Transfer the samples into a 96 well deep well plate Add 200 uL MSL Buffer Vortex to mix thoroughly Incubate at 70 C for 10 minutes Place the plate on the bench for 5 minutes to bring to room temperature Add 260 uL of 100 ethanol 10 uL Mag Bind
21. ed DNA to a nuclease free 1 5 mL microcentrifuge tube Note The expected yield from a 20 mg sample is 8 35 ug genomic DNA depending on type of tissue Store DNA at 20 C Mag Bind Tissue DNA Protocols Mag Bind Tissue DNA Kit Protocol Cultured Cells This protocol is designed for rapid isolation of up to 25 ug genomic DNA from up to 5 x 10 cultured cells Materials and Equipment to be Supplied by User Magnetic separation device for 1 5 mL microcentrifuge tubes Cat MSD 02 e Tabletop microcentrifuge Nuclease free 1 5 mL microcentrifuge tubes Water bath incubator or heat block capable of 70 C e Shaking water bath capable of 55 C Vortexer 100 ethanol Cold PBS 4 C Before Starting e Prepare all Reagents according to Preparing Reagents section on Page 4 Preheat Elution Buffer to 70 C Set shaking water bath to 55 C 1 Prepare the cell suspension la Frozen cell samples should be thawed before starting this protocol Pellet the cells by centrifugation wash the cells with cold PBS 4 C and resuspend cells in 180 uL cold PBS Proceed with Step 2 of this protocol 1b For cells grown in suspension pellet 5 x 10 cells at 1 200 x gina microcentrifuge tube Discard the supernatant and wash the cells once with cold PBS 4 C Resuspend cells in 180 uL cold PBS Proceed with Step 2 of this protocol 1c For cells grown in a monolayer harvest the cells by either using a trypsin tre
22. ed at room temperature for 12 months e Proteinase Storage Buffer is no longer included in the kit Kit Contents Purifications 200 Mag Bind Particles SC SPM Wash Buffer Elution Buffer Proteinase K Solution Purifications Storage and Stability Most components of the Mag Bind Tissue DNA Kit and the Mag Bind Tissue DNA 96 Kit can be stored at room temperature except the Mag Bind Particles SC and LPA Buffer Mag Bind Particles SC and LPA Buffer must be stored at 2 8 C Proteinase K Solution can be stored at room temperature for 12 months For long term storage gt 12 months store Proteinase K Solution at 2 8 C Under these conditions performance of all components of the kit are guaranteed at least 12 months Under cool ambient conditions a precipitate may form in the MSL Buffer and TL Buffer In case of such an event heat the bottle to 37 C to dissolve the precipitate 3 Preparing Reagents 1 Dilute SPM Wash Buffer with 100 ethanol as follows and store at room temperature M6223 02 140 mL M6229 01 175 mL per bottle 2 Dilute MP Buffer with 100 ethanol as follows and store at room temperature M6229 00 37 5 mL M6229 01 150 mL Mag Bind Tissue DNA Protocols Mag Bind Tissue DNA Kit Protocol Tissue This method allows genomic DNA isolation from up to 20 mg tissue Yields vary depending on the source Materials and Equipment to be Supplied by User Magnetic separation device for 1 5 m
23. g Bind Particles SC are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Repeat Steps 20 25 for a second SPM Wash Buffer wash step Leave the tube on the magnetic separation device for 10 minutes to air dry the Mag Bind Particles SC Remove any residual liquid with a pipettor Remove the tube from the magnetic separation device Add 50 200 uL Elution Buffer preheated to 70 C Resuspend the Mag Bind Particles SC by vortexing for 3 minutes or pipetting up and down 50 times Let sit at room temperature for 5 10 minutes 19 32 33 34 20 Mag Bind Tissue DNA Protocols Place the tube on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a nuclease free 1 5 mL microcentrifuge tube Store DNA at 20 C Mag Bind Tissue DNA 96 Protocols Mag Bind Tissue DNA 96 Kit Protocol Tissue This method allows genomic DNA isolation from up to 10 mg tissue Yields will vary depending on the source Materials and Equipment to be Supplied by User Magnetic separation device for 96 well plates Cat MSD 01B or MSD 01 e 96 well deep well plates compatible with the magnetic separation device 96 well microplates e Centrifuge with swing bucket rotor capable of 4000
24. ifuge tubes Cat MSD 02 e Tabletop microcentrifuge e Nuclease free 1 5 mL microcentrifuge tubes Water bath incubator or heat block capable of 70 C Shaking water bath capable of 55 C Vortexer 100 ethanol Before Starting Prepare all Reagents according to Preparing Reagents section on Page 4 Preheat Elution Buffer to 70 C Set shaking water bath to 55 C 1 Cut off the buccal brush or swab head and place into a 1 5 mL microcentrifuge tube 2 Add 400 uL TL Buffer 3 Add 20 uL Proteinase K Solution Vortex to mix thoroughly 4 Incubate at 55 C in a shaking water bath for 45 minutes Note If a shaking water bath is not available vortex the sample every 5 10 minutes 5 Centrifuge at 3 000 x g for 10 minutes 6 Transfer 200 uL lysate into a new 1 5 mL microcentrifuge tube Do not transfer the swab to the new tube 7 Add 200 uL MSL Buffer Vortex or pipet up and down to mix thoroughly 17 10 11 12 13 14 15 16 17 18 18 Mag Bind Tissue DNA Protocols Incubate at 70 C for 10 minutes Place the sample on the bench for 5 minutes to bring the sample to room temperature Add 260 uL 100 ethanol 10 uL Mag Bind Particles SC and 10 uL LPA Buffer Vortex or pipet up and down 20 30 times to mix thoroughly Let sit at room temperature for 5 minutes Place the tube on a magnetic separation device to magnetize the Mag Bind Particles SC Let sit for 10 15 min
25. plete lysis may significantly reduce DNA yields Incubation time for complete tail lysis is dependent on length of tail snip and age of animal e g a 5 mm tail piece from a 2 week old mouse typically will lyse in 2 hours For older animals an overnight incubation may improve yields Note that bone and hair will not lyse Centrifuge at maximum speed for 5 minutes to pellet undigested tissue debris and hair Carefully transfer 200 uL of the supernatant to a new 96 well deep well plate without disturbing the undigested pellet OPTIONAL Mouse tail tissue contains RNA that can purify with the DNA This will not interfere with PCR reactions but other enzymatic reactions may be affected To remove RNA add 5 uL RNase A and let sit at room temperature for 2 minutes 7 10 11 12 13 30 Add 200 uL MSL Buffer Vortex to mix thoroughly Incubate at 70 C for 10 minutes Place the plate on the bench for 5 minutes to bring to room temperature Add 260 uL of 100 ethanol 10 uL Mag Bind Particles SC and 10 uL LPA Buffer Mix thoroughly by vortexing or pipetting Let sit at room temperature for 5 minutes Place the plate on a magnetic separation device to magnetize the Mag Bind Particles SC Let sit for 10 15 minutes Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC 14 15 16 17 18 19 20 21 22 Mag Bind Tissue DNA 96 Protocols Remove the plate from
26. pplied by User Magnetic separation device for 96 well plates Cat MSD 01B or MSD 01 96 well deep well plates compatible with the magnetic separation device e 96 well microplates e Centrifuge with swing bucket rotor capable of 4000 x g e Centrifuge adaptor for 96 well plates Water bath incubator or heat block capable of 70 C Shaking water bath capable of 55 C Vortexer 100 ethanol e Sealing film e Multi channel reservoir Before Starting Prepare all Reagents according to Preparing Reagents section on Page 4 Preheat Elution Buffer to 70 C Set water bath to 55 C 1 Snip a 2 5 mm piece of mouse tail cut into several pieces and transfer the pieces to a 96 well deep well plate Note Follow all regulations regarding the safe and humane treatment of animals Mice should not be older than 6 weeks since lysis will be more difficult in older animals resulting in suboptimal DNA yields If possible obtain tail biopsies at 2 4 weeks and freeze samples at 70 C until DNA is extracted 2 Add 250 uLTL Buffer 3 Add 20 uL of Proteinase K Solution Vortex to mix thoroughly Note Seal the plate before vortexing to prevent cross contamination or loss of sample 29 Mag Bind Tissue DNA 96 Protocols Incubate the plate at 55 C in a shaking water bath for 1 4 hours or until lysis is complete Note If a shaking water bath is not available vortex the samples vigorously every 20 30 minutes Incom
27. s at 2 4 weeks and freeze samples at 70 C until DNA is extracted 2 Add 250 uLTL Buffer 3 Add 20 uL Proteinase K Solution Vortex to mix thoroughly 4 Incubate at 55 C in a shaking water bath for 1 4 hours or until lysis is complete Note If a shaking water bath is not available vortex the samples vigorously every 20 30 minutes Incomplete lysis may significantly reduce DNA yields Incubation time for complete tail lysis is dependent on length of tail snip and age of animal e g a 5 mm tail piece from a 2 week old mouse typically will lyse in 2 hours For older animals an overnight incubation may improve yields Note that bone and hair will not lyse 13 5 Mag Bind Tissue DNA Protocols Centrifuge at maximum speed for 5 minutes to pellet undigested tissue debris and hair Carefully transfer 200 uL of the supernatant to a new 1 5 mL microcentrifuge tube without disturbing the undigested pellet OPTIONAL Mouse tail tissue contains RNA that can be purified with the DNA This will not interfere with PCR reactions but other enzymatic reactions may be affected To remove RNA add 5 uL RNase A and let sit at room temperature for 2 minutes Proceed to Step 6 6 10 11 12 13 14 14 Add 200 uL MSL Buffer Vortex or pipet up and down to mix thoroughly Incubate at 70 C for 10 minutes Place the sample on the bench for 5 minutes to bring the sample to room temperature Add 260 uL 100 ethanol 10 uL Mag
28. the magnetic separation device Add 400 uL MP Buffer Note MP Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles SC by vortexing or pipetting up and down 20 30 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles SC Let sit for 3 minutes at room temperature Vortex or pipet up and down a few times during incubation Place the plate on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Remove the plate from the magnetic separation device Add 400 uL SPM Wash Buffer Note SPM Wash Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles SC by vortexing or pipetting up and down 20 30 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles SC 31 23 24 25 26 27 28 29 30 31 32 33 34 32 Mag Bind Tissue DNA 96 Protocols Let sit for 3 minutes at room temperature Vortex or pipet up and down a few times during incubation Place the plate on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind
29. ticles SC Remove the tube from the magnetic separation device Add 400 uL SPM Wash Buffer Note SPM Wash Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles SC by vortexing or pipetting up and down 20 30 times Let sit for 3 minutes at room temperature Vortex or pipette up and down a few times during incubation Place the tube on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Repeat Steps 19 24 for a second SPM Wash Buffer wash step 26 27 28 29 30 31 32 33 Mag Bind Tissue DNA Protocols Leave the tube on the magnetic separation device for 10 minutes to air dry the Mag Bind Particles SC Remove any residual liquid with a pipettor Remove the tube from the magnetic separation device Add 50 200 uL Elution Buffer preheated to 70 C Resuspend the Mag Bind Particles SC by vortexing for 3 minutes or pipetting up and down 50 times Let sit at room temperature for 5 10 minutes Place the tube on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution Transfer the cleared supernatant containing purifi
30. utes Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Remove the tube from the magnetic separation device Add 400 uL MP Buffer Note MP Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles SC by vortexing or pipetting up and down 20 30 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles SC Let sit for 3 minutes at room temperature Vortex or pipet up and down a few times during incubation Place the tube on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Mag Bind Particles SC are completely cleared from solution 19 20 21 22 23 24 25 26 27 28 29 30 31 Mag Bind Tissue DNA Protocols Aspirate and discard the supernatant Do not disturb the Mag Bind Particles SC Remove the tube from the magnetic separation device Add 400 uL SPM Wash Buffer Note SPM Wash Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles SC by vortexing or pipetting up and down 20 30 times Let sit for 3 minutes at room temperature Vortex or pipet up and down a few times during incubation Place the tube on the magnetic separation device to magnetize the Mag Bind Particles SC Let sit at room temperature until the Ma
31. y genomic DNA from a wide variety of tissues and cultured cells Up to 20 mg animal tissue 1 5 mL microcentrifuge tube format 10 mg animal tissue 96 well deep well plate format or 5 x 10 cells can be processed in less than 1 hour The Mag Bind paramagnetic particles technology provides high quality DNA which is suitable for direct use in most downstream applications such as amplifications and enzymatic reactions This system can be easily adapted to a variety of automated platforms and the procedure can be scaled up or down allowing for purification from various amounts of starting material If using the Mag Bind Tissue DNA Kit or the Mag Bind Tissue DNA 96 Kit for the first time please read this booklet to become familiar with the procedure and its various modifications Animal tissue or cultured cells are pretreated with Proteinase K and lysed in a specially formulated buffer containing detergent DNA binds to the surface of Mag Bind Particles SC Proteins polysaccharides and cellular debris are efficiently washed with three wash steps Pure DNA is eluted in water or low ionic strength buffer Purified DNA can be directly used in downstream applications without the need for further purification 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 step to resuspend prior to use Proteinase K Solution can be stor
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