Mag-Bind®Universal Pathogen 96 Kit - Omega Bio-Tek
Contents
1. Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles RQ Remove the plate containing the Mag Bind Particles RQ from the Magnetic Separation Device Repeat Steps 19 23 once for a second VHB Wash step Add 600 uL SPM Wash Buffer to each sample Resuspend the Mag Bind Particles RQ by vortexing or pipetting up and down 20 times Note SPM Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Let sit at room temperature for 2 minutes 15 27 28 29 30 31 32 33 34 16 Mag Bind Universal Pathogen 96 Kit Urine Protocol Place the 96 well plate on the Magnetic Separation Device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles RQ Leave the plate on the Magnetic Separation Device Add 500 uL nuclease free water not provided to each sample Immediately aspirate the nuclease free water Do not let the samples stay in contact with the nuclease free water for more than 60 seconds Note If using an automated platform use the maximum volume the tips will allow up to 600 UL Add 50 100 uL Elution Buffer heated to 70 C to each samp2. store at room temperature M4029 00 112 mL M4029 01 112 mL per bottle Mag Bind Universal Pathogen 96 Kit Tissue Protocol Mag Bind Universal Pathogen 96 Kit Tissue Protocol Materials and Equipment to be Supplied by User Centrifuge capable of at least 3 500 x g with adaptor for 96 well plates Magnetic Separation Device Recommend Cat AlpAqua 96M A000250 Incubator capable of 70 C 96 well plates with a capacity of at least 1 7 mL Recommend Nunc 278752 and compatible with the Magnetic Separation Device 96 well microplates for DNA storage Vortexer 100 ethanol Nuclease free water Optional Mixer mill such as a SPEX CertiPrep Geno Grinder 2010 or Qiagen TissueLyser Before Starting Prepare VHB Buffer and SPM Wash Buffer according to the Preparing Reagents section on Page 4 Set an incubator to 70 C Heat Elution Buffer to 70 C Briefly spin the E Z 96 Disruptor Plus Plate to remove any glass beads from the walls of the wells Uncap the E Z 96 Disruptor Plus Plate and save the caps for use in Step 3 Add 25 30 mg tissue to each well Add 525 uL SLX Mlus Buffer to each sample Seal the E Z 96 Disruptor Plus Plate with the caps removed in Step 1 Vortex at maximum speed for 3 5 minutes to lyse and homogenize samples For best results a Mixer Mill such as Spex CertiPrep Geno Grinder 2010 or Qiagen Tissuelyser should be used Note Depending on the sample amount and type the amount of SLX M
3. 50 uL DS Buffer and 20 uL Proteinase K Solution to each sample Seal the E Z 96 Disruptor Plus Plate with new Caps for Racked Microtubes provided Vortex for 60 seconds to mix thoroughly Incubate at 70 C for 15 minutes Mix once during incubation Centrifuge at 3 500 x g for 10 minutes Transfer 300 uL cleared supernatant to a 96 well deep well plate 1 7 mL compatible with the Magnetic Separation Device used Note Do not transfer any debris as it can reduce yield and purity Add 300 uL XP2 Buffer 300 uL RBB Buffer and 20 uL Mag Bind Particles RQ to each sample Vortex to mix thoroughly or pipet up and down 20 times Note Mag Bind Particles RQ and XP2 Buffer can be prepared as a mastermix prior to use Prepare only what is needed Tip mixing is recommended for automated protocols for best yield Let sit at room temperature for 10 minutes Place the 96 well deep well plate on the Magnetic Separation Device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles RQ 17 18 19 20 21 22 23 24 25 26 27 Mag Bind Universal Pathogen 96 Kit Serum amp Stool Protocol Remove the plate containing the Mag Bind Particles RQ from the Magnetic Separation Device Add 600 uL VHB Buffer to each sample Resuspend the Ma
4. Ca OMEGA Innovations in nucleic acid isolation bio tek Product Manual Mag Bind Universal Pathogen 96 Kit M4029 00 1 x 96 preps M4029 01 4 x 96 preps August 2015 For research use only Not intended for diagnostic testing Mag Bind Universal Pathogen 96 Kit Table of Contents Introduction and OVEFVIEW csssssssssecseecssccnsecssceseceseessecsnccssecseeesesseeese Kit Contents Storage and Stability essecssssscssecsseesseeessessnseesees Preparing EACLE IIS scpssessesssastisisssaciscesnedtpsecovadcastvancoraciontdencenretoetuoreiaacseance Tissue PECLOCO acssuansustsnnisinemsesnsiniieansenntaandumd cuss an Serum Stool PROC CG sccccksccincchccssceesssecenasee ssnacsssctastchasnceceasssbessaceeantenstaned Urine Protocoles tesistir tae areas aen a Si Troubleshooting Guide ic csnciisssmutsanicudseeanisanisaiacaneins Manual Revision August 2015 N OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview Introduction The Mag Bind Universal Pathogen 96 Kit allows rapid and reliable isolation of high quality host genomic DNA gram positive and negative bacterial DNA fungal spore DNA and viral DNA and viral RNA from tissue urine serum and fecal samples The system allows for automation after sample lysis via Hamilton STAR Thermo KingFisher Flex Applied Biosystems MagMAX Qiagen BioSprint and other liquid handling instruments The system combines the Mag Bind technolo
5. Recommend Nunc 278752 and compatible with the Magnetic Separation Device 96 well microplates for DNA storage Vortexer 100 ethanol Nuclease free water e Optional Mixer mill such as a SPEX CertiPrep Geno Grinder 2010 or Qiagen TissueLyser Before Starting e Prepare VHB Buffer and SPM Wash Buffer according to the Preparing Reagents section on Page 4 Set an incubator to 70 C Heat Elution Buffer to 70 C 1 Briefly spin the E Z 96 Disruptor Plus Plate to remove any glass beads from the walls of the wells Uncap the E Z 96 Disruptor Plus Plate and save the caps for use in Step 3 2 Add 250 uL serum or stool samples to each well If stool sample is solid resuspend to 10 wgt volume in PBS before starting 3 Add 275 uL SLX Mlus Buffer to each sample Seal the E Z 96 Disruptor Plus Plate with the caps removed in Step 1 4 Vortex at maximum speed for 3 5 minutes to lyse and homogenize samples For best results a Mixer Mill such as Spex CertiPrep Geno Grinder 2010 or Qiagen Tissuelyser should be used Note Depending on the sample amount and type the amount of SLX Mlus Buffer may need to be adjusted so that 300 uL can be recovered after Step 5 10 11 12 13 14 15 16 10 Mag Bind Universal Pathogen 96 Kit Serum amp Stool Protocol Centrifuge at 1 000 2 000 x g for 60 seconds at room temperature Remove and discard the caps from the E Z 96 Disruptor Plus Plate Add
6. ansfer 300 uL cleared supernatant to a 96 well deep well plate 1 2 mL compatible with the Magnetic Separation Device used Note Do not transfer any debris as it can reduce yields and purity Add 300 uL XP2 Buffer 300 uL RBB Buffer and 20 uL Mag Bind Particles RQ to each sample Vortex to mix thoroughly or pipet up and down 20 times Note Mag Bind Particles RQ and XP2 Buffer can be prepared as a mastermix prior to use Prepare only what is needed Tip mixing is recommended for automated protocols for best yield Let sit at room temperature for 10 minutes Place the 96 well deep well plate on the Magnetic Separation Device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution 17 18 19 20 21 22 23 24 25 26 Mag Bind Universal Pathogen 96 Kit Urine Protocol Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles RQ Remove the plate containing the Mag Bind Particles RQ from the Magnetic Separation Device Add 600 uL VHB Buffer to each sample Resuspend the Mag Bind Particles RQ by vortexing or pipetting up and down 20 times Note VHB Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Let sit at room temperature for 2 minutes Place the 96 well deep well plate on the Magnetic Separation Device to magnetize the Mag Bind
7. d SPM Wash Buffer according to the Preparing Reagents section on Page 4 Set an incubator to 70 C Heat Elution Buffer to 70 C Prepare an ice Bucket 1 Briefly spin the E Z 96 Disruptor Plus Plate to remove any glass beads from the walls of the wells Uncap the E Z 96 Disruptor Plus Plate and save the caps for use in Step 3 2 Add 250 uL urine sample to each well 3 Add 275 uL SLX Mlus Buffer to each sample Seal the E Z 96 Disruptor Plus Plate with the caps removed in Step 1 4 Vortex at maximum speed for 3 5 minutes to lyse and homogenize samples For best results a Mixer Mill such as Spex CertiPrep Geno Grinder 2010 or Qiagen Tissuelyser should be used Note Depending on the sample amount and type the amount of SLX Mlus Buffer may need to be adjusted so that 300 uL can be recovered after Step 5 13 10 11 12 13 14 15 16 14 Mag Bind Universal Pathogen 96 Kit Urine Protocol Centrifuge at 1 000 2 000 x g for 60 seconds at room temperature Remove and discard the caps from the E Z 96 Disruptor Plus Plate Add 50 uL DS Buffer and 20 uL Proteinase K Solution to each sample Seal the E Z 96 Disruptor Plus Plate with new Caps for Racked Microtubes provided Vortex for 60 seconds to mix thoroughly Incubate at 70 C for 15 minutes Mix once during incubation Add 200 uL PCP Buffer to each well Place the plate on ice for 5 minutes Centrifuge at 3 500 x g for 10 minutes Tr
8. each sample Immediately aspirate the nuclease free water Do not let the samples stay in contact with the nuclease free water for more than 60 seconds Note If using an automated platform use the maximum volume the tips will allow up to 600 uL Add 50 100 uL Elution Buffer heated to 70 C to each sample Resuspend the Mag Bind Particles RQ by vortexing or pipetting up and down 20 times Let sit at room temperature for 5 minutes Place the 96 well plate on the Magnetic Separation Device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a clean 96 well microplate Store the DNA at 20 C Mag Bind Universal Pathogen 96 Kit Urine Protocol Mag Bind Universal Pathogen 96 Kit Urine Protocol Materials and Equipment to be Supplied by User Centrifuge capable of at least 3 500 x g with adaptor for 96 well plates Magnetic Separation Device Recommend Cat AlpAqua 96M A000250 Incubator capable of 70 C 96 well plates with a capacity of at least 1 7 mL Recommend Nunc 278752 and compatible with the Magnetic Separation Device 96 well microplates for DNA storage Vortexer 100 ethanol Nuclease free water e Optional Mixer mill such as a SPEX CertiPrep Geno Grinder 2010 or Qiagen TissueLyser e Ice Bucket Before Starting e Prepare VHB Buffer an
9. ed off Make sure that water wash step does not exceed 60 seconds and the Mag Bind Particles RQ are not resuspended Water Wash extended After water is added during wash step Mag Bind Particles Mag Bind Particles RQ will go into solution RQ lost in process Let magnetic beads remagnetize prior to aspirating liquid BSA not added to Add BSA to a final concentration of 0 1 ug mL PCR mixture to the PCR mixture Too much DNA Dilute the DNA elute used in the downstream inhibits PCR reactions application if possible Non specific bands in Use hot start Taq polymerase mixture downstream PCR Ipay i Inhibitory substance Check the A A ratio 260 230 in the eluted DNA Dilute the elute to 1 50 if necessary 17
10. g Bind Particles RQ by vortexing or pipetting up and down 20 times Note VHB Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Let sit at room temperature for 2 minutes Place the 96 well deep well plate on the Magnetic Separation Device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles RQ Remove the plate containing the Mag Bind Particles RQ from the Magnetic Separation Device Repeat Steps 18 22 once for a second VHB Wash step Add 600 uL SPM Wash Buffer to each sample Resuspend the Mag Bind Particles RQ by vortexing or pipetting up and down 20 times Note SPM Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Let sit at room temperature for 2 minutes Place the 96 well plate on the Magnetic Separation Device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles RQ 11 28 29 30 31 32 33 12 Mag Bind Universal Pathogen 96 Kit Serum amp Stool Protocol Leave the plate on the Magnetic Separation Device Add 500 uL nuclease free water not provided to
11. gy with RBB Buffer to eliminate PCR inhibiting compounds within the samples Purified DNA is suitable for PCR restriction digestion and hybridization applications There are no organic extractions thus reducing plastic waste and hands on time and multiple samples can be processed in parallel Kit Contents Product Number M4029 00 M4029 01 Purifications 1x96 preps 4x96 preps E Z 96 Disruptor Plus Plates Caps for Racked Microtubes Mag Bind Particles RQ 9mL SLX Mlus Buffer 240 mL DS Buffer 30 mL PCP Buffer 100 mL XP2 Buffer 160 mL RBB Buffer 160 mL VHB Buffer 3 x 88 mL SPM Wash Buffer 4x 30mL Elution Buffer 50 mL Proteinase K Solution 9mL User Manual Storage and Stability All of the Mag Bind Universal Pathogen 96 Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows Mag Bind Particles RQ must be stored at 2 8 C Proteinase K Solution can be stored at room temperature for up to 12 months For long term storage store Proteinase K Solution at 2 8 C All remaining components should be stored at room temperature During shipment or storage in cool ambient conditions precipitates may form in some of the buffers Dissolve such deposits by warming the solution at 37 C and gently shaking Preparing Reagents 1 Dilute SPM Wash Buffer with 100 ethanol as follows and store at room temperature M4029 01 70 mL per bottle 2 Dilute VHB Buffer with 100 ethanol follows and
12. he Mag Bind Particles RQ 17 18 19 20 21 22 23 24 25 26 27 Mag Bind Universal Pathogen 96 Kit Tissue Protocol Remove the plate containing the Mag Bind Particles RQ from the Magnetic Separation Device Add 600 uL VHB Buffer to each sample Resuspend the Mag Bind Particles RQ by vortexing or pipetting up and down 20 times Note VHB Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Let sit at room temperature for 2 minutes Place the 96 well deep well plate on the Magnetic Separation Device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles RQ Remove the plate containing the Mag Bind Particles RQ from the Magnetic Separation Device Repeat Steps 18 22 once for a second VHB Wash step Add 600 uL SPM Wash Buffer to each sample Resuspend the Mag Bind Particles RQ by vortexing or pipetting up and down 20 times Note SPM Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Let sit at room temperature for 2 minutes Place the 96 well plate on the Magnetic Separation Device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution A
13. le Resuspend the Mag Bind Particles RQ by vortexing or pipetting up and down 20 times Let sit at room temperature for 5 minutes Place the 96 well plate on the Magnetic Separation Device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a clean 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 Problem Aj co Aoaq ratio 3 230 is low A A ratio 260 280 is high Low DNA Yield or no DNA Yield Problems in downstream applications Problems in downstream applications Repeat the DNA isolation with a new sample Extend the incubation time with VHB Buffer Wash the Mag Bind Particles RQ with ethanol Salt contamination The protocol does not remove RNA If desired add 5 uL RNase A 25 mg mL after lysate is cleared and before binding buffers are added Let sit at room temperature for 5 minutes RNA contamination Repeat the DNA isolation with a new sample be sure to mix the sample with SLX Mlus Buffer thoroughly Use a commercial homogenizer if possible Make sure VHB Buffer and SPM Wash Buffer are mixed with ethanol Poor homogenization of sample DNA wash
14. lus Buffer may need to be adjusted so that 300 uL can be recovered after Step 5 10 11 12 13 14 15 16 Mag Bind Universal Pathogen 96 Kit Tissue Protocol Centrifuge at 1 000 2 000 x g for 60 seconds at room temperature Remove and discard the caps from the E Z 96 Disruptor Plus Plate Add 53uL DS Buffer and 20 uL Proteinase K Solution to each sample Seal the E Z 96 Disruptor Plus Plate with new Caps for Racked Microtubes provided Vortex for 60 seconds to mix thoroughly Incubate at 70 C for 15 minutes Mix once during incubation Centrifuge at 3 500 x g for 10 minutes Transfer 300 uL cleared supernatant to a 96 well deep well plate 1 7 mL compatible with the Magnetic Separation Device used Note Do not transfer any debris as it can reduce yield and purity Add 300 uL XP2 Buffer 300 uL RBB Buffer and 20 uL Mag Bind Particles RQ to each sample Vortex to mix thoroughly or pipet up and down 20 times Note Mag Bind Particles RQ and XP2 Buffer can be prepared as a mastermix prior to use Prepare only what is needed Tip mixing is recommended for automated protocols for best yield Let sit at room temperature for 10 minutes Place the 96 well deep well plate on the Magnetic Separation Device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb t
15. spirate and discard the cleared supernatant Do not disturb the Mag Bind Particles RQ 7 28 29 30 31 32 33 Mag Bind Universal Pathogen 96 Kit Tissue Protocol Leave the plate on the Magnetic Separation Device Add 500 uL nuclease free water not provided to each sample Immediately aspirate the nuclease free water Do not let the samples stay in contact with the nuclease free water for more than 60 seconds Note If using an automated platform use the maximum volume the tips will allow up to 600 uL Add 50 100 uL Elution Buffer heated to 70 C to each sample Resuspend the Mag Bind Particles RQ by vortexing or pipetting up and down 20 times Let sit at room temperature for 5 minutes Place the 96 well plate on the Magnetic Separation Device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a clean 96 well microplate Store the DNA at 20 C Mag Bind Universal Pathogen 96 Kit Serum amp Stool Protocol Mag Bind Universal Pathogen 96 Kit Serum amp Stool Protocol Materials and Equipment to be Supplied by User Centrifuge capable of at least 3 500 x g with adaptor for 96 well plates Magnetic Separation Device Recommend Cat AlpAqua 96M A000250 Incubator capable of 70 C 96 well plates with a capacity of at least 1 7 mL
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