Manual - Omega Bio-Tek
Contents
1. 1 Freeze and grind up to 500 mg of arthropod tissue under liquid nitrogen Grind tissue completely to obtain a fine homogenous powder 2 Immediately add 2 5 ml of Buffer RB 2 mercaptoethanol Add 20ul of 2 mercaptoethanol per 1ml of Buffer RB and then add 2 5 ml of this mixture to the sample Samples should not be allowed to thaw before Buffer RB 2 mercaptoethanol is added Vortex vigorously to make sure that all clumps are dispersed RNA cannot be effectively extracted from clumped tissue Note Add 20ul of 2 mercaptoethanol per 1 ml of Buffer RB before use This mixture can be made and stored at room temperature for 1 week 3 Proceed with the Plant RNA Midi prep Protocol from step 3 page 4 RNA Isolation from Fungi E Z N A Plant RNA Kit can also be used for fungal RNA isolation since many fungal samples possess similar cellular attributes as many plant specimens 1 Freeze and grind up to 500 mg of fungal sample under liquid nitrogen Grind tissue completely to obtain a fine homogenous powder 2 Immediately add 2 5 ml Buffer RB 2 mercaptoethanol Add 10ul of 2 mercaptoethanol per 1ml of Buffer RB and then add 2 5ml of this mixture to the sample Samples should not be allowed to thaw before Buffer RB 2 mercaptoethanol is added Vortex vigorously to make sure that all of the clumps are dispersed RNA cannot be effectively extracted from clumped tissue 3 Proceed with the Plant RNA Protocol from step 3 page 4 Quantiza2. back into the collection tube Add 3 5 ml of Wash Buffer II diluted with ethanol Centrifuge at 3 000 5 000 x g for 30 seconds at room temperature Discard the flow through and place the column back into the collection tube Wash the column with a second 3 5 ml of Wash Buffer Il as in previous step Centrifuge and discard the flow through Then with the collection tube empty centrifuge the Midi spin column at 3 000 5 000 x g for 10 minutes to completely dry the HiBind matrix Elution of RNA Transfer the column to a RNase free 15 ml microfuge tube not supplied and elute the RNA with 500yl of DEPC treated water supplied Make sure to add water directly onto column matrix Centrifuge at 6000 x g for 5 minutes at room temperature A second elution into the same tube may be necessary if the expected yield of RNA gt 300 yg DNase digestion Protocol Optional Since HiBind RNA resin and spin column technology actually removes most of DNA without the DNase treatment it is not necessary to do DNase digestion for most downstream applications However certain sensitive RNA applications might require further DNA removal The following steps provide on membrane DNase digestion see DNase Cat E1091 for further information 1 Follow the standard protocol until the samples have completely passed through the HiBind RNA column Prepare the following A Pipet 1 5ml of RNA Wash Buffer into the HiBind RNA Midi spin column a
3. 93 01 02 E Z N A Total RNA Maxi Kit Total RNA isolation from animal cells or tissues R6615 01 02 E Z N A Blood RNA Midi Kit Total RNA isolation from blood samples R6616 01 02 E Z N A Blood RNA Maxi Kit Total RNA isolation from blood samples Total RNA isolation from plant samples mRNA isolation Single reagent for total RNA isolation R6248 01 02 E Z N A RNA Probe purification kit RNA Probe purification R6249 01 02 R6376 01 02 E Z N A Poly Gel RNA Isolation Kit Isolate RNA from poly acrylamide gel l R6500 01 02 E Z N A Oligo dT Cellulose High capacity oligo dT cellulose RNase free DNase set R6628 01 02 E Z N A Plant RNA Midi Kit Other RNA isolation kit Reagent and supplies R6511 01 02 mRNA Enrichment kit or ceo R6830 01 02 RNA Solv reagent DNase set for on column DNase digestion Page 12 of 12
4. Contents l trod ctioM ache niet ete ee wate tet dw day at de ee Ede ae bee eae 2 New In This Edition 2 0 nee 2 Storage and Stability 0 0 0 0 000 ee 2 Binding Capacity 0 000 2 RIP Contents aaam deine Qarinady aaa aa a 4 Gala a Bence Rad eee ee ee BRS 3 Before Statins s cacciac eeecacakiee ae ae oie pee eels ae RE eee 3 Working WHR RNA 22 2 2 ecee eee teh tes ee es be ee CR ES 3 E Z N A Plant RNA Midiprep Protocol standard protocol 4 E Z N A Plant RNA Midiprep Protocol for difficultsamples 6 On Membrane DNase Digestion 020220 02 eee 8 RNA Isolation from Arthropods 0000 00 c eee 9 RNA Isolation from Fungal 00000 0c eee eee eee 10 RNA Quality ossea nis anaa a angie ie ge ed Oe ial ee a gee eS 10 Quantization and Storage of RNA 0 0 ee ee 10 Troubleshooting Guide 0 0 cee eee 11 Ordering Information 000000 cece eee 12 Introduction The E Z N A Plant RNA Midiprep Kit provides a convenient and rapid method for the isolation of total RNA from a variety of plant samples The kit includes shredding homogenizing units to efficiently remove cell debris and simultaneously homogenize the lysate In combination with HiBind RNA spin columns this permits purification of high quality RNA from as much as 500 mg of tissue Typical yields are shown in Table 1 E Z N A Plant RNA Kits are ideal for processing mult
5. Ensure not to introduce RNase during the procedure Check buffers for RNase contamination Ensure Wash Buffer II has been diluted with 100 ethanol as indicated on bottle Diluted Wash Buffer II must be stored at room temperature Repeat wash with Wash Buffer II Digest with RNase free DNase and inactivate at 75 C for 5 min DEPC treated water is acidic and can dramatically lower Abs260 values Use TE buffer pH 8 to dilute RNA prior to spec analysis Ordering Information Product Number E Z N A Total RNA Miniprep Kits Product Name Description R6634 01 02 R6834 01 02 E Z N A Total RNA Kit Total RNA isolation from animal cells or tissues R6614 01 02 E Z N A Blood RNA Kit R6814 01 02 Total RNA Isolation from blood samples R6627 01 02 R6827 01 02 E Z N A Plant RNA Kit Total RNA Isolation from plant samples R6640 01 02 E Z N A Fungal RNA Kit R6840 01 02 samples Total RNA Isolation from fungal R6670 01 02 E Z N A Yeast RNA Kit R6870 01 02 Total RNA Isolation from yeast samples R6850 01 02 R6950 01 02 R6675 01 02 ZNA Mollusc RNA Kit Total RNA Isolation from mollusc R6875 01 02 invertebrates samples E Z N A Bacterial RNA Kit Total RNA Isolation from yeast samples E Z N A Total RNA Midi maxi Kits R6664 01 02 E Z N A Total RNA Midi Kit Total RNA isolation from animal cells or tissues R66
6. ds commonly found in plant tissues Use this protocol when standard protocol did not yield RNA or got lower yield 1 Grind plant sample as described on page 4 Collect frozen ground plant tissue up to 500 mg in a microfuge tube and immediately add 3 5ml of Buffer RPL 2 mercaptoethanol We recommend starting with 250 mg of tissue at first If results obtained are satisfactory increase amount of starting material Add 20ul of 2 mercaptoethanol per 1ml of Buffer RPL Samples should not be allowed to thaw before Buffer RPL 2 mercaptoethanol is added Vortex vigorously to make sure that all of the clumps are dispersed RNA cannot be effectively extracted from clumped tissue Note Add 20ul of 2 mercaptoethanol per 1 ml of Buffer RPL before use This mixture can be made and stored at room temperature for 1 week 2 Add 700ul of Buffer SP and vortex thoroughly to mix Centrifuge at 3 000 5 000 x g for 20 minutes at room temperature 3 Carefully aspirate cleared lysate to a RNase free 15ml centrifuge tube making sure not to disturb the pellet or transfer any debris Add one volume of isopropanol and vortex to precipitate RNA This step removes much of the polysaccharide content and improves spin column performance by increasing RNA binding capacity and therefore yield in the steps that follow No incubation is required after addition of isopropanol TIP In most cases 3 5 ml supernatant can easily be removed This will require 3 5 ml isopropano
7. euse the collection tube Note Wash Buffer Il Concentrate must be diluted with absolute ethanol before use Refer to label on bottle for directions 4 Wash column with a second 3 5 ml of Wash Buffer Il by repeating step 3 Centrifuge and discard the flow through 5 Then with the collection tube empty centrifuge the Midi spin column at 3 000 5 000 x g for 10 minutes at full speed to completely dry the HiBind matrix 6 Elution of RNA Transfer the column to a clean 15 ml microfuge tube not supplied and elute the RNA with 50 100 ul of DEPC treated water supplied Make sure to add water directly onto the column matrix Centrifuge for 1 minute at maximum speed A second elution may be necessary if the expected yield of RNA gt 50 ug Alternatively RNA may be eluted with a greater 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 water to 70 C before adding to column and incubating column for 5 minutes at room temperature before centrifugation may increase yields RNA Isolation from Arthropods The exoskeleton of arthropods poses the same problems as encountered with many plant specimens Pigments and polysaccharides often co purify with nucleic acids and interfere with downstream applications Page 8 of 12 Prepare allnecessary materials and reagents listed on page 4 and follow the procedure below
8. iple 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 RT PCR Northern Analysis Differential display e e e e Poly A RNA selection Yields obtained with E Z N A Plant RNA Kits Arabidopsissp O O O Arabidopsis sp 150 ug Tobacco leaves O O OOOO leaves 200 ug Mustard leaves O OOOO leaves 150 Pra L ow f ow f Storage and Stability All components of the E Z N A 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 conditions a precipitate may form in the Buffer RB In case of such an event heat the bottle at 37 C to dissolve Store Buffer RB at room temperature Binding Capacity Each HiBind RNA column can bind approximately100ug of RNA Using greater than 200 mg of plant tissue usually will not dramatically improve yields and sometimes has adverse effects Page 2 of 12 Kit Contents i E Z N A Plant RNA Protocol I Standard Protocol Product No R6628 00 R6628 01 R6628 02 ae Materials to be provided by user TM HiBind i RNA columns 2 10 25 e Swing Bucket centrifuge capable of 3 000 5 000 x g 15 ml Collection Tubes 4 20 50 e Nuclease free 15ml conical centrifuge tubes Homogenizer Midi Colu
9. l Note that depending on the sample the volume of supernatant may vary After transferring to a fresh tube measure the volume and add the correct amount of isopropanol 4 Immediately centrifuge at 3 000 5 000 x g for 20 min at room temperature to pellet RNA A longer centrifugation does not improve yields 5 Carefully aspirate or decant the supernatant and discard making sure not to dislodge the RNA pellet Invert the microfuge tube on a paper towel for 5 min to allow residual liquid to drain Drying the pellet is not necessary 6 Add 500u of RB buffer pre heated to 65 C and vortex to resuspend the pellet A brief incubation at 65 C may be necessary to effectively dissolve the RNA 7 Add 1 25ml of Buffer RB 2 mercaptoethanol followed by 1 75ml of 70 ethanol Vortex thoroughly to mix This will adjust binding conditions prior to loading the HiBind RNA column Page 6 of 12 10 11 12 13 Apply the entire sample including any precipitates that may form to an HiBind RNA Midi spin column assembled in a clean 15 ml collection tube supplied Centrifuge at 3 000 5 000 x g for 5 minutes at room temperature Discard the flow through liquid and place the column back into the collection tube Note This is the starting point to perform DNase digestion See page 8 for detail protocol Add 3 5ml of RNA Wash Buffer and centrifuge at 3 000 5 000 x g for 5 minutes Discard the flow through liquid and place the column
10. membrane DNase digestion treatment or after elution DNase digestion will be needed For modified protocols for DNase digestion call our technical staff at 800 832 8896 for assistance Page 10 of 12 Troubleshooting Guide Problem Little or no RNA RNA remains eluted on the column Column is overloaded Clogged column Incomplete disruption or lysis of plant tissue Precipitated RNA High nucleic will not dissolve acid and polysaccharide content Degraded RNA RNase contamination Problem in Salt carry over downstream during elution applications DNA contamination Co purification of DNA Low Abs ratios RNA diluted in acidic buffer or water Suggestion Repeat elution Pre heat DEPC water to 70 C prior to elution Incubate column for 10 min with water prior to centrifugation Reduce the quantity of starting material Completely disrupt sample in liquid nitrogen Increase centrifugation time Reduce amount of starting material Reduce the amount of starting material Generally it is best to start with 50 100 mg at first To avoid RNA degradation do not increase incubation time for resuspension Freeze starting material quickly in liquid nitrogen and store at 70 C without thawing Follow protocol closely and work quickly Make sure that 2 mercaptoethanol is added to Buffer RPL Use RB Buffer as dissolvent instead of DEPC water
11. mns 2 10 25 e 2 mercaptoethanol e Absolute 96 100 ethanol Buffer RPL 10 ml 40ml 100 ml l e Isopropyl alcohol isopropanol Buffer RB 6 ml RNA Wash Buffer II Concentrate 5 ml This protocol is suitable for most fresh or frozen tissue samples allowing DEPC treated water 1 5 ml 20 ml 30 ml l efficient recovery of RNA However due to the tremendous variation in water and polysaccharide content of plants sample size should be limited User Manual 1 1 1 to lt 500 mg Best results are obtained with young leaves or needles SSS SS SS SS SS SS Buffer RB contains a chaotropic salt Use gloves and protective eye ware when handling this solution e Liquid nitrogen for freezing disrupting samples e Preheat an aliquot 500 ul per sample of DEPC treated water at 65 C Note Use extreme caution when handling liquid nitrogen Note that all centrifugation steps must be carried out at room temperature Before Starting Please take a few minutes to read this booklet thoroughly to become familiar with the 1 Weigh up to 500mg of plant sample Immediately place the protocol Prepare all materials required before starting to minimize RNA degradation weighed sample in liquid nitrogen and grind thoroughly with a mortar and pestle Decantthe powder and liquid nitrogen into an Rnase free liquid nitrogen cooled 50mI centrifuge tube not Dilute Wash Buffer II with absolute ethanol as follows supplied Allow the liquid nit
12. nd centrifuge at 3 000 5 000 x g for 5 minutes to wash the column Discard the flow through For each HiBind RNA Midi spin column prepare the DNase digestion reaction mix as follows OBI DNase Digestion Buffer 367 5 ul i RNase free DNase 20 Kunitz unites ul 7 5 ul Total volume 375 ul Note 1 DNase lis very sensitive and prone to physical denaturing so do not vortex the DNase mixture Mix gently by inverting the tube Prepare the fresh DNase digestion mixture before RNA isolation 2 OBI DNase digestion buffer is supplied with OBI RNase free Dnase set 3 Standard DNase buffers are not compatible with on membrane Dnase digestion B Pipet 375 ul of the DNase digestion reaction mix directly onto the surface of the HiBind RNA matrix in each column Make sure to pipet the Dnase digestion mixture directly onto the membrane Dnase digestion will not be complete if some of the mix sticks to the wall or the O ring of the HiBind RNA column C Incubate at room temperature 25 30 C for 15 minutes 2 Place column into a clean 15 0ml collection tube and add 3ml of RNA Wash Buffer I Incubate for 5 minutes at room temperature Centrifuge at 6000 x g for 2 minutes and discard the flow through Reuse the collection tube 3 Place the column into the same 15ml collection tube and add 3 5ml of RNA Wash Buffer II diluted with ethanol Centrifuge at 3 000 5 000 x g for 5 minutes and discard the flow through R
13. r 1 ml of Buffer RB or RPL This 4 Carefully transfer the supernatant of the flow through fraction to mixture can be stored for 1 week at room temperature anew 15 ml centrifuge tube making sure not to disturb the pellet Page 4 of 12 or transfer any debris Add 0 5 volume of absolute ethanol and mix by vortexing TIP In most cases 2 0 ml supernatant can easily be removed This will require 1 0 ml ethanol Note that depending on the sample the volume of supernatant may vary After transferring to a fresh tube measure the volume and add the correct amount of ethanol 5 Apply the entire sample including any precipitates that may form to a HiBind RNA Midi Spin column assembled in a 15 0 ml collecting tube supplied Centrifuge at 3 000 5 000 x g for 5 minutes at room temperature Discard the flow through liquid and place the column back into the collection tube Optional on membrane DNase digestion This is the starting point to perform DNase digestion See Page 8 for detailed protocol 6 Add 3 5 ml RNA Wash Buffer I Centrifuge at 3 000 5 000 x g for 5 minutes Discard the flow through liquid and place the column back into the collection tube 7 Add 3 5ml Wash Buffer Il diluted with ethanol Centrifuge at 3 000 5 000 x g for 2 minutes at room temperature Then discard the flow through and place the column back into the collection tube Note Wash Buffer II Concentrate must be diluted with absolute ethanol before use Refe
14. r to the label on the bottle for directions 8 Wash column with a second 3 5ml of Wash Buffer Il by repeating previous step Centrifuge and discard the flow through 9 Then with the collection tube empty centrifuge the Midi spin column for 10 min at 3 000 5 000 x g to completely dry the HiBind matrix 11 Elution of RNA Transfer the column to a new RNase free 15 ml centrifuge tube not supplied and elute the RNA with 500uI of DEPC treated water supplied Make sure to add water directly onto column matrix Centrifuge at 3 000 5 000 x g for 5 minutes A second elution into the same tube may be necessary if the expected yield of RNA gt 300 ug Note RNA may be eluted with a greater 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 No RNA extraction procedure can completely remove genomic DNA For sensitive work such as RT PCR or differential display we suggest that you treat the eluted RNA with RNase free DNase Also for RT PCR use intron spanning primers that allow easy identification of DNA contamination E Z N A Plant RNA Midi Protocol Il for difficult samples Certain plant samples are very difficult for RNA isolation because of amount of material and type of secondary metabolites This method involves a simple and rapid precipitation step for removal of much of the polysaccharides and phenolic compoun
15. rogen to evaporate but do not IMPORTANT allow the sample to thaw R6628 00 Add 20ml 100 ethanol R6628 01 Add 48 mI 100 ethanol 2 Immediately add 2 5ml of Buffer RB 2 mercaptoethanol We R6628 02 Add 200 mI 100 ethanol recommend starting with 250 mg of tissue at first If results obtained are satisfactory increase amount of starting material Add 25ul of 2 mercaptoethanol per 2 5 ml of Buffer RB Samples should not be Working with RNA allowed to thaw before Buffer RB 2 mercaptoethanol is added Vortex vigorously to make sure that all of the clumps are dispersed RNA cannot be effectively extracted from clumped tissue e Whenever working with RNA always wear latex gloves to minimize RNase contamination Change gloves frequently Use only clean RNase free TIP As a guide a 2 cm diameter leaf square weighs approximately disposable plastic pipette tips when using the supplied reagents 100 mg e During the procedure work carefully but quickly P abati 3 Transfer the lysate directly into a Homogenization Midi Spin e Under cool ambient conditions crystals may form in Buffer RB This is Column placed in the collection tube Centrifuge at 3 000 5 000 x normal and the bottle may be warmed to redissolve the salt g for 15 minutes at room temperature e 2 mercaptoethanol R mercaptoethanol is key in denaturing endogenous RNases and must be added to an aliquot of Buffer RB and Buffer RPL before use Add 20ul of 2 mercaptoethanol pe
16. tion and Storage of RNA To determine the concentration and purity of RNA measure absorbancy at 260 nm and 280 nm in a spectrophotometer 1 O D unit measured at 260 nm corresponds to 40 ug of RNA per ml The ratio of A o A of pure nucleic acids is 2 0 while for pure protein it is approximately 0 6 A ratio of 1 8 2 0 corresponds to 90 100 pure nucleic acid Phenol has an absorbancy maximum at 275 nm and can interfere with spectrophotometric analysis of DNA or RNA However the E Z N A Plant RNA Kit eliminates the use of phenol and avoids this problem Store RNA samples at 70 C in water Under such conditions RNA prepared with the E Z N A system is stable for more than a year RNA Quality It is highly recommended that RNA quality be determined prior to all analyses The quality of RNA can be assessed by denaturing agarose gel electrophoresis and ethidium bromide staining Several sharp bands should appear on the gel These are the 28S and 18S ribosomal RNA bands as well as certain populations of mRNA and possibly viral RNA bands If these bands smear towards lower molecular weight RNAs then the RNA has undergone major degradation during preparation handling or storage RNA molecules less than 200 bases in length do not efficiently bind the HiBind matrix thus the method enriches high quality RNA Since no RNA extraction procedure can completely remove genomic DNA For sensitive work such as RT PCR or differential display either on
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