Manual - Omega Bio-Tek
Contents
1. RT PCR qPCR e Northern Analysis e Differential Analysis e Poly A RNA Selection Binding Capacity Each well of the HiBind RNA plate can bind approximately 50 ug RNA Using more than the recommended maximum amount of plant tissue usually will not improve yields significantly and often has adverse effects New In this Edition e The latest edition has been newly designed to enhance readability and protocol quality 2 mL Collection Plates are now called 96 well Square well Plates This is a name change only there has been no change to the plastic ware Illustrated Centrifugation Protocol Grind and Lyse Sample Transfer Cleared Lysate to a Homogenizer Plate Adjust Binding Conditions Bind RNA and Wash 3X Dry The RNA Plate Elute Kit Contents Product No R1027 02 E Z 96 RNA Plate 96 well Square well Plate 2 2 ml eS SS ee e296 homogenizer gt a 96 well Racked Microtubes 1 2 ml Caps for Racked Microtubes 96 well Square well Plate 2 2 ml are reusable see Page 5 for instructions RB Buffer and RNA Wash Buffer 1 contains a chaotropic salt Use gloves and protective eye ware when handling this solution Storage and Stability All components of the E Z 96 Plant RNA Kit should be stored at 22 C 25 C Under these conditions RNA has successfully been purified and used for RT PCR after 24 months of storage Under cool ambient cond2. and discard flow through Re use the collection plate Place the HiBind RNA Plate on top of the same 96 well Square well Plate and add 700 ul RNA Wash Buffer II diluted with ethanol Centrifuge at 4 000 x g for 3 minutes and discard flow through Re use the collection plate Note RNA Wash Buffer Il Concentrate must be diluted with absolute ethanol before use Refer to Page 3 or to the bottle label for directions Optional DNase Digestion Protocol 4 Add 700ul of RNA Wash Buffer II diluted with ethanol Centrifuge at 4 000 x g for 3 minutes Discard flow through and re use the collection plate 5 Centrifuge the empty HiBind RNA Plate at 4 000 x g for 10 min to completely dry the HiBind matrix Complete drying is critical 6 Elution of RNA Place the HiBind RNA Plate on top of the 96 well Racked Microtubes supplied Add 100 ul of DEPC Water supplied with kit to each well Make sure to add DEPC Water directly to the center of each membrane for optimal elution results Incubate at room temperature for 1 minute Centrifuge for 5 min at 4 000 x g to elute RNA Alternatively RNA may be eluted with a greater or lesser volume of water While additional elutions increase total RNA yield the concentration will be lowered since more than 80 of RNA is recovered with the first elution Pre heating the DEPC Water to 65 C before adding to the HiBind RNA Plate and incubating the plate for 5 min at room temperature before centrifugation may
3. E Z 96 Plant RNA Kit Table of Contents Introd tiONssisiuntusiuniusmsintn nnn noiai 2 Mistral ais 3 Kit Contents Storage and Stability 4 Preparing Reagents Cleaning Plates 5 Quantification of RNA ueaesssesneneneenennnennennennennnennenn 6 E Z 96 Plant RNA Spin Protocol eenee 7 Optional DNase Digestion Protoco 10 Troubleshooting Guide 12 O RIRRTUIRRES RSERR NER REITER NEON FEANRLENNERNURSEERRRELRUEHSRENER 14 Manual Revision August 2012 Fi OMEGA bio tek Innovations in nucleic acid isolation Introduction The E Z 96 Plant RNA Kit provides a convenient and rapid method for the isolation of total RNA from a variety of plant samples The kit includes a homogenization plate to efficiently remove cell debris and simultaneously homogenize the lysate In combination with the HiBind RNA plate this kit permits purification of high quality RNA from as much as 40 mg seed tissue or as much as 100 mg plant tissue The system is efficient enough to allow isolation of total RNA from as little as 0 5 mg tissue Typical yields are shown in Table 1 E Z 96 Plant RNA Kits are ideal for processing multiple plant samples in less than one hour The need for organic extractions is eliminated making total RNA isolation fast safe and reliable Purified RNA has Abs260 Abs280 ratios of 1 8 2 0 and is suitable for the following applications
4. are well Plate supplied making sure not to disturb the pellet or transfer any debris Add 0 5 volume absolute ethanol and mix by vortexing Keep and re use the 2 ml collection plate for Step 6 Tip In most cases 450 ul supernatant can easily be removed This will require 225ul ethanol The volume of supernatant may vary For convenience a fixed volume may be used Measure the volume and add the correct amount of ethanol Apply the entire sample including any precipitates that may form to a HiBind RNA Plate placed on top of the 96 well Square well Plate from previous step Seal the plate with aera seal film Centrifuge at 4 000 x g for 5 minutes at room temperature Discard the flow through liquid and place the HiBind RNA Plate back on top of the collection plate If the sample volume exceeds the well capacity load successively and repeat this step Note Be sure that the lysate has passed completely through each well If any lysate remains repeat centrifugation for an additional 3 to 5 minutes DNase I Digestion OPTIONAL This is the point to begin optional DNase I digestion If DNase digestion is necessary for downstream applications go to Page 6 to complete the procedure using the Dnase Digestion Protocol otherwise continue with the next step Add 600 ul RNA Wash Buffer I into each well of the HiBind RNA Plate Seal the plate with aera seal film Then centrifuge at 4 000 x g for 5 minutes Discard the flow through liquid and pla
5. ce the HiBind RNA Plate into a New 2 mL Collection Plate 10 11 E Z 96 Plant RNA Spin Protocol Add 700 pl RNA Wash Buffer Il diluted with ethanol Seal the plate with aera seal film and centrifuge at 4 000 x g for 5 minutes at room temperature Discard the flow through and re use the collection plate in next step Add 700 pl RNA Wash Buffer Il diluted with ethanol Seal the plate with aera seal film and centrifuge at 4 000 x g for 5 minutes at room temperature Discard the flow through and re use the collection plate in next step Centrifuge the HiBind RNA Plate for 10 min at 4 000 x g to completely dry the HiBind matrix Elution of RNA Place the HiBind RNA Plate on top of the 96 well Racked Microtubes supplied Add 100 ul of DEPC Water supplied with kit to each well Make sure to add DEPC Water directly to the center of each membrane for optimal elution results Incubate at room temperature for 1 minute Centrifuge for 5 min at 4 000 x g to elute RNA Note RNA may be eluted with a greater or lesser volume of water While additional elutions increase total RNA yield the concentration will be lowered since more than 80 of RNA is recovered with the first elution Optional DNase Digestion Protocol DNase Digestion Protocol Optional Since the HiBind RNA technology eliminates most of the DNA DNase digestion is not necessary for most downstream applications However certain sensitive RNA applications might requi
6. e the Plates from the GenoGrinder Carefully remove the caps Add 500 ul RB Buffer 2 mercaptoethanol per sample to the wells Vortex the samples to disperse the clumps Procede to Step 3 E Z 96 Plant RNA Spin Protocol Add 500 ul RB Buffer 2 mercaptoethanol per sample to the wells of a 2 ml deep well plate Note Add 10 ul 2 mercaptoethanol per 1 ml of RB Buffer before use This mixture can be made and stored at room temperature for 1 month 2 mercaptoethanol should be added again estimate same proportion if RB Buffer is stored for more than 1 month Collect frozen ground plant tissue up to 100 mg or seed tissue up to 30 mg and add to a well containing RB Buffer 2 mercaptoethanol Samples should not be allowed to thaw before adding to RB Buffer 2 mercaptoethanol We recommend starting with 30 to 50 mg plant tissue or 12 to 20 mg seed tissue If results obtained are satisfactory increase amount of starting material up to maximum limits Vortex vigorously to make sure that all clumps are dispersed RNA cannot be effectively extracted from clumped tissue Tip Asa guide a 2 cm diameter leaf square weighs approximately 100 mg Centrifuge the plate at 4 000 x g for 10 minutes Pipet the lysate directly into a E Z 96 Well Homogenizer Plate placed on top of a new 2 ml collection plate supplied Centrifuge at 3 500 x g for 10 min at room temperature Carefully transfer the supernatant of the flow through fraction to a new 96 well Squ
7. horoughly with tap water incubate overnight in 0 2M NaOH 1mM EDTA rinse with distilled water and dry by air Quantification of RNA Storage of RNA Purified RNA can be stored at 20 C or 70 C RNase free water Under such conditions RNA prepared with the E Z N A Plant RNA Kit is stable for more than a year Quantification of RNA To determine the concentration and purity of RNA one should measure the absorbance at 260 nm and 280 nm in a spectrophotometer 1 O D unit measured at 260 nm corresponds to 40ug of RNA per ml DEPC Water is slightly acidic and can dramatically lower absorbance values We suggest that you dilute the sample in a buffered solution TE for spectrophotometric analysis The A A ratio of pure nucleic acids is 2 0 while for pure protein is approximately 0 6 Therefore a ratio of 1 8 2 0 corresponds to 90 100 pure nucleic acid Phenol has a maximum absorbance at 275 nm and can interfere with absorbance readings of DNA or RNA However the E Z N A Plant RNA Kit eliminates the use of phenol and avoids this problem RNA Quality Itis highly recommended that RNA Quality be determined prior to all analysis The quality of RNA can be best assessed by denaturing agarose gel electrophoresis and ethidium bromide staining Two sharp bands should appear on the gel These are the 285 and the 185 23S and 165 for bacteria ribosomal RNA bands If these band appears as a smear towards lower molecular weight sized RNA
8. increase yields 11 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 800 832 8896 Possible Problems and Suggestions Repeat elution step RNA remains on the Little or no RNA Membrane Pre heat DEPC Water to 70 C prior to eluted elution Membrane is overloaded Reduce quantity of starting material Completely homogenize sample Clogged Incomplete SORN Increase centrifugation time Membrane homogenization g Reduce amount of starting material Freeze starting material quickly in liquid nitrogen Starting Tissue Do not store tissue prior to extraction un Problems less they are lysed first Degraded RNA Follow protocol closely and work quickly Ensure not to introduce RNase during the procedure RNase contamination Check buffers for RNase contamination 12 Troubleshooting Guide Problem Ensure RNA Wash Buffer II Concentrate has been diluted with 4 volumes of 100 ethanol as indicated on bottle Problem in downstream applications Salt carry over during 1 X RNA Wash Buffer Il must be stored and elution used atroom temperature Repeat wash with RNA Wash Buffer II DNA contamination DEPC Water is acidic and can dramatically RNA diluted in acidic lower Abs260 values Use TE buffer to buffer or water dilute RNA prior to spectrophotometric analysis DNA contamination Digest
9. itions a precipitate may form in the RB Buffer The crystals may be dissolved by heating the buffer at 37 C and gently shaking its container Store RB Buffer and all other components at room temperature Preparing Reagents Dilute RNA Wash Buffer II with absolute ethanol 96 100 as follows a Ethanol To Be Added R1027 00 Add 140 mL absolute ethanol R1027 01 Add 140 mL absolute ethanol R1027 02 Add 200 mL absolute ethanol per bottle Note Itis not necessary to DEPC treat the absolute ethanol before adding to Wash Buffer Il Whenever working with RNA always wear latex gloves to minimize RNase contamination Change gloves frequently Use only clean RNase free disposable plastic pipette tips when using the supplied reagents During the procedure work carefully but quickly Under cool ambient conditions crystals may form in RB Buffer This is normal and the bottle may be warmed to redissolve the salt 2 mercaptoethanol mercaptoethanol is key in denaturing endogenous RNases and must be added to an aliquot of RB Buffer before use Add 10 ul of 2 mercaptoethanol per 1 ml of RB Buffer This mixture can be stored for 1 month at room temperature If RB Buffer is stored for more than 1 month 2 mercaptoethanol should be added again Cleaning of 96 Well Collection Plates If extra plates are needed please call our customer service department for ordering information To re use the 96 Well collection plates rinse them t
10. r and polysaccharide content of plants sample size should be limited to lt 100 mg plant tissue and lt 40 mg seed tissue Less starting material often results in better quality yields Best results are obtained with young leaves or needles This method isolates sufficient RNA for a few tracks on a standard Northern assay depending on the type and quality of the sample Wearing latex disposable gloves collect tissue in a 1 5 ml or 2 ml microfuge tube and freeze by dipping in liquid nitrogen with a pair of tweezers to fill the tube Grind the tissue using disposable pestles available from OBI Cat SSI 1014 39 amp 1015 39 or equivalent Alternatively one can allow liquid nitrogen to evaporate and then store samples at 70 C for later use Do not allow samples to thaw Use disposable pestles only once Alternatively a small clean mortar and pestle can be used Optional Homogenization Using Genogrinder 2000 2010 Add one 3 4 mm stainless steel bead to each well of a 96 well round well plate compatiable with a Genogrinder Cryoadaptor and the plant samples Close the individual tubes with Cap Strips Freeze the sample in liquid nitrogen in the CryoBlock Station 2600 and Cryoadaptor according to Manufacturer s instructions Place the The racks or plates are fixed into clamps on a homogenizer Homogenize for 2 Minutes at 1750 RPM Tissue samples are disrupted and simultaneously homogenized with the shearing and crushing action of the beads Remov
11. re further DNA removal Follow the steps below for on membrane DNase digestion see DNase Digestion Set Cat E1091 or E1091 02 for further information 10 Note DNase is very sensitive and prone to physical denaturing do not vortex the DNase mixture Mix gently by inverting the tube Prepare the fresh DNase digestion mixture before beginning the RNA isolation procedure Standard DNase buffers may not be compatible with Omega Bio Tek s DNase Digestion Set Follow the standard protocol until the samples completely pass through the HiBind RNA Plate Steps 1 6 Then complete the procedure using the following steps A Add 300ul RNA Wash Buffer to each well of the HiBind RNA Plate and centrifuge at 4 000 x g for 1 min B For each RNA sample prepare the DNase digestion mixture as follows E Z N A DNase Digestion Buffer 73 5ul RNase Free DNase 20 Kunitz ul 1 5ul Total Volume 75ul C Pipet 75 ul DNase digestion mixture directly onto the surface of the membrane in each well of the HiBind RNA Plate Be certain to pipet the mixture directly onto each membrane as DNA digestion might not be complete if some of the mixture is retained on the walls or the O rings of the HiBind RNA Plate D Incubate at room temperature 15 30 C for 15 minutes Place the HiBind RNA Plate on top of a clean 96 well Square well Plate and add 400 pl RNA Wash Buffer I Incubate 3 minutes at room temperature Centrifuge at 4 000 x g
12. s the it is likely that your RNA has undergone major degradation during preparation handling or storage Although RNA molecules less than 200 bases in length do not efficiently bind to the HiBind matrix a third RNA band the tRNA band may be visible when a large amount of sample is used Expected Yields Table 1 shows sample yields obtained using the standard protocol under normal conditions Sample size 100 mg Table 1 Yields obtained with E Z N A Plant RNA Kits Arabidopsis sp 30 ug E Z 96 Plant RNA Spin Protocol E Z 96 Plant RNA Protocol Note that all centrifugation steps must be carried out at room temperature Materials to be provided by user Centrifuge capable of 4 000 x g Centrifuge rotor adaptor for 96 well microplates Multichannel pipet RNase free filter pipette tips Racked RNase free 1 2 ml microtubes 2ml 96 well deep well plate 2 mercaptoethanol Absolute 96 100 ethanol Isopropyl alcohol isopropanol Liquid nitrogen for freezing disrupting samples Water bath or heat block preset at 65 C Optional Genogrinder 2000 2010 with CryoBlock 2600 and CryoAdaptor 2650 Before Starting Preheat an aliquot 100 ul per sample of DEPC Water to 65 C Set a Water Bath or Heat Block to 65 C Note Use extreme caution when handling liquid nitrogen This protocol is suitable for most fresh or frozen tissue samples allowing efficient recovery of RNA However due to the tremendous variation in wate
13. with RNase free DNase Low Abs ratios 13 14 Ordering Information The following components are available for purchase separately Call Toll Number 1 800 832 8896 DEPC Water 100 mL RNase free DNase Set 50 preps RNase free DNase Set 200 preps E Z 96 Homogenizer Plates 4x 96 96 well Microplates 500 uL Hibind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc Qiagen QlAvac and Vacman are all trademarks of their respected companies PCR is a patented process of Hoffman La Roche Use of PCR process requires a license Notes Notes
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