Manual - Omega Bio-Tek
Contents
1. microplate not provided Store RNA at 80 C 15 Troubleshooting Guide Please use this guide to solve any problems that may arise We hope that it will aid in clearing up any questions for you If for any reason you need further assistance please contact our technical support staff at our Toll Free Number 1 800 832 8896 Possible Problems and Suggestions Incomplete resuspension Resuspend the Mag Bind Particles CNR of Mag Bind Particles CNR PY vortexing before use Make sure the sample is properly stored and make sure the samples are processed immediately after collection or removal from storage Loss of Mag Bind Particles Be careful not to remove the Mag Bind CNR during procedure Particles CNR during the procedure Ethanol was not added to Add ethanol to RNA Wash Buffer II as RNA Wash Buffer II instructed on Page 4 RNA degraded during sample storage Low RNA yields Mag Bind Particles CNR not resuspended during binding Problem with downstream application Insufficient RNA was used Carryover of the Mag Bind Particles CNR in the elution Carryover of the Mag Bind Particles CNR in the eluted RNA will not effect 16 downstream applications Vortex vigorously for 2 minutes after addition of ethanol and Mag Bind Particles CNR RNA in the sample already degraded Do not freeze thaw the sample more than once Do not store at room temperature To remove the carryover Mag Bind P2. Bind Particles CNR by vortexing or pipetting up and down 20 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles CNR 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 Repeat Steps 22 25 for a second RNA Wash Buffer II wash step Leave the plate on the magnetic separation device for 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 Note All liquid must be aspirated at this step It is helpful to remove all liquid from the well then wait one minute and remove and residual liquid from the well Add 50 100 uL RNA Elution Buffer to each sample Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Note Complete resuspension is required for efficient elution 32 33 34 Mag Bind Total RNA 96 Kit Protocols 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 Transfer the cleared supernatant containing purified RNA to a clean 96 well
3. Note The Mag Bind Particles CNR will settle and clump together at the bottom of the bottle during storage Vortex the Mag Bind Particles CNR thoroughly before use Mag Bind Particles CNR and ethanol can be made as a mastermix Vortexing vigorously for 60 seconds is critical for obtaining good yield Let sit for 5 minutes at room temperature 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 VHB Buffer to each sample Note VHB Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles CNR 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 Mag Bind Total RNA 96 Kit Protocols 14 Add 400 uL RNA Wash Buffer II to each sample Note RNA Wash Buffer II must be
4. medium completely Add 450 uL TRK Lysis Buffer and 20 uL Proteinase K Solution to each sample Pipet up and down several time to mix the samples Trypsinization of cells Aun PWN gt 7 8 9 10 11 12 Determine the number of cells Aspirate the cell culture medium completely Wash cells with 4 C PBS Aspirate the PBS Wash cells with 4 C PBS containing 0 1 0 25 trypsin Check cells for detachment Make sure cells are detached before proceeding Add cell culture medium containing serum to inactivate the trypsin Transfer cells to an RNase free microplate Centrifuge at 500 x g for 5 minutes Aspirate the supernatant completely Add 450 uL TRK Lysis Buffer and 20 uL Proteinase K Solution to each sample Pipet up and down several time to mix the samples Note Not removing cell culture medium completely will inhibit lysis and dilute the lysate This will affect the conditions for binding of RNA to the Mag Bind Particles CNR Proteinase K Solution and TRK Lysis Buffer can be added as a freshly prepared mastermix Centrifuge at 4 000 x g for 5 minutes Transfer 400 uL lysate to a 96 well plate Do not disturb the debris pellet Note The 96 well plate must have minimum volume of 1 0 mL and be compatible with the magnetic stand used 11 10 11 12 13 12 Mag Bind Total RNA 96 Kit Protocols Add 300 uL 100 ethanol and 20 uL Mag Bind Particles CNR to each sample Vortex for 60 seconds
5. Mag Bind Total RNA 96 Kit Table of Contents Introducti ON eiea E ES 2 Kit Contents Storage and Stability cesssecsccssecseccneersees 3 Preparing Reagents ssesseessssesseeesssseceesssseeceessseeecossseeeoosesssee 4 Mag Bind Total RNA 96 Protocol TiSSUe sssssssssesssssse 5 Mag Bind Total RNA 96 Protocol Cultured Cells 10 Troubleshooting Guide ssesssssseersssssssesseeeseesssssssseeeseessnsssss 16 Manual Revision April 2012 024 OMEGA bio tek Innovations in nucleic acid isolation Introduction Mag Bind Total RNA 96 Kit allows for rapid and reliable isolation of high quality total cellular RNA from a wide variety of tissue and cultured cells Total RNA can be purified from 5 10 mg of tissue or 1 x 10 of cultured cells Purified RNA is suitable for all major downstream applications such as RT PCR restriction digestion and hybridization applications If using the Mag Bind Total RNA 96 Kit for the first time please read this booklet in its entirety to become familiar with the procedures Samples are lysed in TRK Lysis Buffer Proteins are removed with a Proteinase K digestion step DNA and RNA are then bound to the paramagnetic beads Genomic DNA is removed with a DNase digestion step After two quick wash steps with 70 Ethanol RNA is eluted Since the Mag Bind Total RNA 96 Kit uses paramagnetic beads it can also be adapted to liquid handlers including Beckman Coutler s Biomek F
6. X Tecan Genesis Thermo Kingfisher Flex and Hamilton Star instruments New in this Edition Proteinase K is now supplied in a liquid form eliminating the step to respuspend 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 Product Number M6731 00 M6731 01 Purifications 1 x 96 preps 4 x 96 preps Mag Bind Particles CNR 2 2 mL 8 8 mL TRK Lysis Buffer 50 mL 200 mL VHB Buffer 22 mL 88 mL RNA Wash Buffer II 50 mL 150 mL Proteinase K Solution 3 mL 12 mL DNase 220 uL 4x 220 uL DNase Digestion Buffer 12 mL 48 mL RNA Elution Buffer 15 mL 50 mL User Manual v Bera Storage and Stability Most components of the Mag Bind Total RNA Kit are stable for at least 12 months from date of purchase when stored at 22 C 25 C Mag Bind Particles Solution should be stored at 2 8 C Dnase 1 Enzyme should be stored at 20 C During shipment or storage in cool ambient conditions precipitates may form in Buffer TRK and VHB Dissolve such deposits by warming the solution at 45 C and gently shaking Preparing Reagents 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 1 Dilute VHB Buffer with 100 ethan
7. articles CNR from the eluted RNA simply place the plate on the magnetic separation device and wait until the eluate has cleared Carefully transfer the RNA eluate to a new 96 well plate
8. diluted with ethanol prior to use Please see Page 4 for instructions 15 Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles CNR 16 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 17 Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Note All liquid must be aspirated at this step It is helpful to remove all liquid from the well then wait one minute and remove and residual liquid from the well 18 Remove the plate from the magnetic separation device 19 Let sit at room temperature for 2 minutes While waiting prepare the DNase mix Amount per Prep Total Amount per 96 well Plate DNase Digestion Buffer 98 uL 10 34 mL A 10 pipetting error has been calculated for a 96 well plate 20 Add 100 uL DNase to each sample Mix by pipetting up and down to fully resuspend the magnetic beads 21 Let sit at room temperature for 10 minutes 13 22 23 24 25 26 27 28 29 30 31 14 Mag Bind Total RNA 96 Kit Protocols Add 400 uL RNA Wash Buffer Il to each sample Note RNA Wash Buffer II must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag
9. ecommend Cat MSD 01B Equipment for disrupting tissue MM300 Mixer Mill or Geno Grinder 2000 2010 or mortar and pestle Vortexer 100 Ethanol lsopropanol Pre chilled PBS if using cells grown in a monolayer Trypsin if using cells grown in a monolayer Multi channel pipettor and nuclease free pipette tips Sealing film Recommend Cat AC1200 01 Multi channel reservoirs Recommend Cat AC1331 01 96 well microplates with a minimum capacity of 1 mL Recommend Cat SSI 1780 00 or Thermo 96 well deep V microplate for Kingfisher Flex Part No 95040450 Before Starting 1 10 Prepare buffers according to instructions on Page 4 Vortex the Mag Bind Particles CNR thoroughly before use Harvest cells by choosing one of the following methods A or B A For cells grown in suspension 1 Determine the number of cells Do not use more than 1 x 10 cells 2 Pellet the appropriate number of cells by centrifuging at 500 x g for 5 minutes 3 Add 450 uL TRK Lysis Buffer and 20 uL Proteinase K Solution to each sample 4 Pipet up and down several time to mix the samples Mag Bind Total RNA 96 Kit Protocols B For cells grown in a monolayer These cells can either be lysed directly in the cell culture dish or trypsinized and collected as a cell pellet prior to lysis Cells grown in cell culture flasks should always be trypsinized Direct cell lysis 1 2 3 4 Determine the number of cells Aspirate the cell culture
10. etic 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 VHB Buffer to each sample Note VHB Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles CNR 12 13 14 13 16 17 18 19 20 Mag Bind Total RNA 96 Kit Protocols 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 RNA Wash Buffer Il to each sample Note RNA Wash Buffer II must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles CNR Place the plate on the magnetic separation device to magnetize
11. lean pestle Transfer the grounded powder and liquid nitrogen into 96 well deep well plate and allow the liquid nitrogen to evaporate Add 450 uL TRK Lysis Buffer B Mechanical Tissue Disruption Place sample into a stainless steel grinding plate with appropriate steel beads Add 450 uL TRK Lysis Buffer to the samples Grind sample at 30 Hz for 1 2 minutes according to manufacturer s instructions For GenoGrinder 2000 2010 grind at 1500 RPM for 2 minutes Remove the plate from the homogenizer and remove the caps It may be necessary to centrifuge the plate briefly to remove debris from the caps 10 11 Mag Bind Total RNA 96 Kit Protocols Add 20 uL Proteinase K Solution to each sample Vortex or invert several times to mix the samples Centrifuge at 4 000 x g for 5 minutes Transfer 400 uL lysate to a 96 well plate Do not disturb the debris pellet Note The 96 well plate must have minimum volume of 1 0 mL and be compatible with the magnetic stand used Add 300 uL 100 ethanol and 20 uL Mag Bind Particles CNR to each sample Vortex for 60 seconds Note The Mag Bind Particles CNR will settle and clump together at the bottom of the bottle during storage Vortex the Mag Bind Particles CNR thoroughly before use Mag Bind Particles CNR and ethanol can be made as a mastermix Vortexing vigorously for 60 seconds is critical for obtaining good yield Let sit for 5 minutes at room temperature Place the plate on the magn
12. ol as follows and store at room temperature M6731 01 112 mL M6731 02 112 mL per bottle 2 Dilute RNA Wash Buffer II with 100 ethanol as follows and store at room temperature M6731 00 200 mL per bottle M6731 01 600 mL per bottle M6731 02 600 mL per bottle Mag Bind Total RNA 96 Kit Protocols Mag Bind Total RNA 96 Kit Protocol Tissue Materials and Equipment to be Supplied by User Centrifuge capable of 4 000 x g with swing bucket rotor for 96 well plates Adapter for 96 well deep well plates Magnetic separation device Recommend Cat MSD 01B Equipment for disrupting tissue MM300 Mixer Mill or Geno Grinder 2000 2010 or mortar and pestle e Vortexer 100 Ethanol e lsopropanol Multi channel pipettor and nuclease free pipette tips Sealing film Recommend Cat AC1200 01 Multi channel reservoirs Recommend Cat AC1331 01 e 96 well microplates with a minimum capacity of 1 mL Recommend Cat SSI 1780 00 or Thermo 96 well deep V microplate for Kingfisher Flex Part No 95040450 Before Starting e Prepare buffers according to instructions on Page 4 Vortex the Mag Bind Particles CNR thoroughly before use 1 Choose one of the two following methods for homogenization of samples A Manual Sample Preparation To prepare samples collect fresh tissue sample in a 30 mL mortar and freeze by dipping in liquid nitrogen using tweezers or tongs to fill the tube Grind the tissue using a c
13. 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 Note All liquid must be aspirated at this step It is helpful to remove all liquid from the well then wait one minute and remove and residual liquid from the well Remove the plate from the magnetic separation device Let sit at room temperature for 2 minutes While waiting prepare the DNase mix Amount per Prep Total Amount per 96 well Plate DNase Digestion Buffer 98 uL 10 34 mL A 10 pipetting error has been calculated for a 96 well plate 21 22 23 24 25 26 27 28 29 30 31 Mag Bind Total RNA 96 Kit Protocols Add 100 uL DNase I to each sample Mix by pipetting up and down to fully resuspend the magnetic beads Let sit at room temperature for 10 minutes Add 400 uL RNA Wash Buffer Il to each sample Note RNA Wash Buffer II must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles CNR 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 sol
14. ution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Remove the plate from the magnetic separation device Repeat Steps 23 26 for a second RNA Wash Buffer II wash step Leave the plate on the magnetic separation device for 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 Note All liquid must be aspirated at this step It is helpful to remove all liquid from the well then wait one minute and remove and residual liquid from the well Add 50 100 uL RNA Elution Buffer to each sample 32 33 34 35 Mag Bind Total RNA 96 Kit Protocols Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Note Complete resuspension is required for efficient elution 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 Transfer the cleared supernatant containing purified RNA to a clean 96 well microplate not provided Store RNA at 80 C Mag Bind Total RNA 96 Kit Protocols Mag Bind Total RNA 96 Kit Protocol Cultured Cells Materials and Equipment to be Supplied by User Centrifuge capable of 4 000 x g with swinging bucket rotor for 96 well plates Adapter for 96 well deep well plates Magnetic separation device R
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