Mag-Bind®Plant RNA Kit Mag
Contents
1. 27 28 29 30 31 32 33 34 35 36 37 38 39 Mag Bind Plant RNA Kit Let sit at room temperature for 5 minutes Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit for 5 minutes or until the sample is clear Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Remove the plate from the magnetic separation device Add 700 uL RNA Wash Buffer Il Vortex at maximum speed to completely resuspend the Mag Bind Particles CNR Let sit at room temperature for 2 minutes Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit for 5 minutes or until the sample is clear Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR 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 Remove the plate from the magnetic separation device Add 100 uL RNA Elution Buffer Seal the plate Vortex at maximum speed to completely resuspend the Mag Bind Particles CNR Let sit at room temperature for 5 minutes Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit for 5 minutes or until the sample is clear Transfer the cleared supernatant containing purified RNA to a new 96 well plate and store the RNA at 70 C 132. Troubleshooting Guide Please use this guide to solve any problems that may arise If for any reason you need further assistance please contact our technical support staff at our Toll Free Number at 1 800 832 8896 Possible Problems and Suggestions Troubleshooting Guide Problem Likely Cause Suggestions Resuspend the magnetic particles Incomplete re suspension by vortexing before use of magnetic particles Make sure the sample is properly RNA degraded during stored and the samples are sample storage processed immediately after Low RNA yields collection or removal from storage VHB Buffer LPX Binding Prepare VHB Buffer LPX Binding Buffer and RNA Wash Buffer Buffer and RNA Wash Buffer II as Il not prepared correctly instructed on Page 4 Loss of magnetic particles Be careful not remove the magnetic during procedure particles during the procedure VHB Buffer and RNA Wash No RNA eluted Buffer Il are not diluted with repare VHB Purr end RNA Wash Buffer Il as instructed on Page 4 100 ethanol RNA in the sample already degraded Do not freeze and thaw the sample more than once Do not store at room temperature too long Problem with downstream Insufficient RNA was used application To remove the carryover magnetic Carryover of Carryover of magnetic particles from the eluted RNA the magnetic particles in the eluted RNA simply magnetize the magnetic particles during will not effect downstream particles and
3. Particles CNR Let sit at room temperature for 5 minutes Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit for 5 minutes or until the sample is clear Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Remove the tube from the magnetic separation device Add 700 uL RNA Wash Buffer Il Vortex at maximum speed to completely resuspend the Mag Bind Particles CNR Let sit at room temperature for 2 minutes Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit for 5 minutes or until the sample is clear Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR 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 from the magnetic separation device Add 100 uL RNA Elution Buffer Vortex at maximum speed to completely resuspend the Mag Bind Particles CNR 37 38 39 Mag Bind Plant RNA Kit Let sit at room temperature for 5 minutes Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit for 5 minutes or until the sample is clear Transfer the cleared supernatant containing purified RNA to a new 1 5 mL microcentrifuge tube and store the RNA at 70 C Mag Bind Plant RNA Kit Mag Bind Plant RNA Protocol
4. 21 22 23 24 25 12 Mag Bind Plant RNA Kit Let sit at room temperature for 2 minutes Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit for 5 minutes or until the sample is clear Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Remove the plate from the magnetic separation device Add 700 uL RNA Wash Buffer II Seal the plate Vortex at maximum speed to completely resuspend the Mag Bind Particles CNR Note RNA Wash Buffer II must be diluted with 100 ethanol before use Please see Page 4 for instructions Let sit at room temperature for 2 minutes Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit for 5 minutes or until the sample is clear Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR 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 Remove the plate from the magnetic separation device Add 200 uL DNase mixture 198 uL DNase Digestion Buffer and 2 uL DNase I Mix thoroughly by pipetting up and down 10 times or inverting the plate 10 times Let sit at room temperature for 15 minutes to digest genomic DNA Add 500 uL RNA Wash Buffer II Seal the plate Vortex at maximum speed to completely resuspend the Mag Bind Particles CNR 26
5. carefully transfer elution applications the RNA eluate to a new microcentrifuge tube 14
6. 96 well Plate Format M6927 Materials and Equipment to be Provided by User e Centrifuge capable of 4 000 x g with swinging bucket rotor for 96 well plates Sealing film 2 2 mL KF Deep well Plate Recommended Cat OME1799 100 ethanol Equipment for disrupting plant tissue MM300 Mixer Mill or Geno Grinder 2000 Mortar and pestle for manual tissue disruption Magnetic stand for 96 well plate Cat MSD 01B Incubator capable of 55 C Before Starting Prepare Buffers according to the Preparing Reagents Section on Page 4 Setan incubator to 55 C Manual sample disruption To prepare samples collect a fresh plant sample in a 30 mL mortar and freeze in liquid nitrogen Grind the tissue using a clean pestle Allow the liquid nitrogen to evaporate and transfer the sample into a 96 well deep well plate Immediately proceed with the RNA isolation protocol below Mechanical tissue disruption Place the plant sample into a stainless steel grinding plate with the appropriate steel beads Freeze the sample in the stainless steel grinding plate for 1 minute with liquid nitrogen Immediately attach the grinding plate onto the clamps of the tissue grinder Grind the sample at 30Hz for 1 2 minutes Immediately proceed with the RNA isolation protocol below 1 Collect frozen ground plant sample start with 30 50 mg in a deep well plate Note Keep the sample frozen until adding RXP Buffer 2 Add 600 uL RXP Buffer Sea
7. Ca OMEGA Innovations in nucleic acid isolation bio tek Product Manual Mag Bind Plant RNA Kit Mag Bind Plant RNA 96 Kit M6828 00 5 preps M6828 01 50 preps M6927 00 1 x 96 preps M6927 01 4x 96 preps July 2013 For research use only Not intended for diagnostic testing Mag Bind Plant RNA Kit Mag Bind Plant RNA 96 Kit Table of Contents LOA WCLION scii ia 2 Kit Contents Storage and Stability 0 ce ceeseeeees 3 Preparing REAGENUS 02 sccsisscsscevssesnectetaseeessnscoundacssnecsonnnrecis 4 Mag Bind Plant RNA Kit Protocol M6828 06 5 Mag Bind Plant RNA 96 Kit Protocol M6927 10 Troubleshooting Guide cccccccscesscssesscseessesseeeeesees 14 Manual Revision July 2013 N OMEGA bio tek Innovations in nucleic acid isolation Introduction The Mag Bind Plant RNA Kit and the Mag Bind Plant RNA 96 Kit allow rapid and reliable isolation of high quality total cellular RNA from a wide variety of plant species and tissues Total RNA from 50 mg of sample can be processed in less than 1 hour The system uses an innovative Mag Bind plant RNA technology to eliminate polysaccharides phenolic compounds and enzyme inhibitors from plant lysate The Mag Bind Plant RNA Kit provides high quality RNA Purified RNA is suitable for all major downstream applications such as RT PCR and microarray analysis Since this kit uses magnetic particles the protocol can be easily ada
8. l the plate Vortex at maximum speed for 1 minute 10 10 11 12 Mag Bind Plant RNA Kit Incubate at 55 C for 3 minutes Centrifuge at maximum speed for 10 minutes to pellet cell debris Note Compact pellets will form in the wells but some particles may float Carefully transfer 400 uL cleared supernatant to a new 2 2 mL KF Deep well Plate Note Do not disturb the pellet or transfer any debris as it may contain proteins and DNA Add 400 uL LPX Binding Buffer Seal the plate Vortex at maximum speed for 30 seconds Note LPX Binding Buffer must be diluted with 100 ethanol before use Please see Page 4 for instructions Add 10 uL Mag Bind Particles CNR and 20 uL Proteinase K Solution Seal the plate Vortex at maximum speed for 30 seconds Let sit at room temperature for 5 minutes Mix the samples a few times during the incubation by gently shaking the plate Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit for 5 minutes or until the lysate is clear Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Remove the plate from the magnetic separation device Add 500 uL VHB Buffer Seal the plate Vortex at maximum speed to completely resuspend the Mag Bind Particles CNR Note VHB Buffer must be diluted with 100 ethanol before use Please see Page 4 for instructions 11 13 14 15 16 17 18 19 20
9. nation Use only clean RNase free disposable plastic pipette tips when using the supplied reagents Materials and Equipment to be Provided by User Centrifuge capable of 13 000 x g Nuclease free 1 5 mL microcentrifuge tubes 100 ethanol Equipment for disrupting plant tissue MM300 Mixer Mill or Geno Grinder 2000 Mortar and pestle for manual tissue disruption Magnetic Separation Device for 1 5 mL microcentrifuge tubes Cat MSD 02 Incubator or water bath capable of 55 C Vortex Before Starting Prepare Buffers according to the Preparing Reagents Section on Page 4 Setan incubator to 55 C Manual sample disruption To prepare samples collect a fresh plant sample in a 30 mL mortar and freeze in liquid nitrogen Grind the tissue using a clean pestle Allow the liquid nitrogen to evaporate and transfer the sample into a 1 5 mL microcentrifuge tube Immediately proceed with the RNA isolation protocol on Page 6 Mechanical tissue disruption Place the plant sample into a stainless steel grinding jar with the appropriate steel beads Freeze the sample in the stainless steel grinding jar for 1 minute with liquid nitrogen Immediately attach the grinding jar onto the clamps of the tissue grinder Grind the sample at 30Hz for 1 2 minutes Immediately proceed with the RNA isolation protocol on Page 6 10 11 Mag Bind Plant RNA Kit Collect frozen ground plant sample start with 30 50 mg in a 1 5 mL microce
10. ntrifuge tube Note Keep the sample frozen until adding RXP Buffer Add 600 uL RXP Buffer Vortex at maximum speed for 30 seconds Incubate at 55 C for 3 minutes Centrifuge at maximum speed for 10 minutes Note Compact pellets will form in the tubes but some particles may float Carefully transfer 400 uL cleared supernatant to a new 1 5 mL microcentrifuge tube Note Do not disturb the pellet or transfer any debris as it may contain proteins and DNA Add 400 uL LPX Binding Buffer Vortex at maximum speed for 30 seconds Note LPX Binding Buffer must be diluted with 100 ethanol before use Please see Page 4 for instructions Add 10 uL Mag Bind Particles CNR and 20 uL Proteinase K Solution Vortex at maximum speed for 30 seconds Let sit at room temperature for 5 minutes Mix the sample a few times during the incubation by gently inverting the tube Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit for 5 minutes or until the lysate is clear Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Remove the tube from the magnetic separation device 12 13 14 15 16 17 18 19 20 21 22 23 Mag Bind Plant RNA Kit Add 500 uL VHB Buffer Vortex at maximum speed to completely resuspend the Mag Bind Particles CNR Note VHB Buffer must be diluted with 100 ethanol before use Please see Page 4 for instr
11. pted to most robotic liquid handling instruments New in this Edition 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 e Proteinase Storage Buffer is no longer included in the kit Kit Contents Storage and Stability Most components of the Mag Bind Plant RNA Kit and the Mag Bind Plant RNA 96 Kit are stable for at least 12 months from date of purchase when stored at 22 25 C Mag Bind Particles CNR should be stored at 2 8 C Proteinase K and DNase should be store at 20 C for long term storage Preparing Reagents Dilute VHB Buffer with 100 ethanol as follows and store at room temperature M6828 01 33 6 mL M6927 00 33 6 mL M6927 01 140 mL Dilute RNA Wash Buffer II with 100 ethanol as follows and store at room temperature S T nae _ Dilute LPX Binding Buffer with 100 ethanol as follows and store at room temperature M6927 01 100 mL Mag Bind Plant RNA Kit Mag Bind Plant RNA Protocol 1 5 mL Tube Format M6828 Please take a few minutes to read this manual thoroughly to become familiar with the protocol before beginning the procedure Prepare all materials required before starting to minimize RNA degradation Wear gloves protective goggles and take great care when working with chemicals Whenever working with RNA always wear gloves to minimize RNase contami
12. uctions Let sit at room temperature for 2 minutes Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit for 5 minutes or until the sample is clear Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Remove the tube from the magnetic separation device Add 700 uL RNA Wash Buffer Il Vortex at maximum speed to completely resuspend the Mag Bind Particles CNR Note RNA Wash Buffer II must be diluted with 100 ethanol before use Please see Page 4 for instructions Let sit at room temperature for 2 minutes Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit for 5 minutes or until the sample is clear Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR 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 from the magnetic separation device Add 200 uL DNase mixture 198 uL DNase Digestion Buffer and 2 uL DNase I Mix thoroughly by pipetting up and down 10 times or inverting the tube 10 times 24 25 26 27 28 29 30 31 32 33 34 35 36 Mag Bind Plant RNA Kit Let sit at room temperature for 15 minutes to digest genomic DNA Add 500 uL RNA Wash Buffer Il Vortex at maximum speed to completely resuspend the Mag Bind
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