5B,B3B.JOJ#&45 6OJWFSTBM3/&YUSBDUJPO,JU … · 4 TaKaRa MiniBEST Universal RNA Extraction Kit Cat. v00Da RL:http: III. Storage and Shipping 1. Part I …

Cat. # 9767

Product Manual

TaKaRa MiniBESTUniversal RNA Extraction Kit

For Research Use

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2

TaKaRa MiniBEST Universal RNA Extraction KitCat. #9767

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URL:http://www.takara-bio.com

Table of ContentsI. Description........................................................................................3

II. KitComponents..............................................................................3

III. StorageandShipping...................................................................4

IV. PrecautionsforPreventingRNaseContamination...........4

V. PrecautionsbeforeStarting......................................................4

VI. SampleAmountandBufferRLVolume.................................5

VII. Protocols............................................................................................6

VIII. ExperimentalExamples..............................................................11

IX. YieldsoftotalRNAfromVariousMaterials.........................12

X. Appendix..........................................................................................12

XI. Troubleshooting...........................................................................13

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TaKaRa MiniBEST Universal RNA Extraction KitCat. #9767v201306Da


I. Description

TaKaRa MiniBEST Universal RNA Extraction Kit is designed for the rapid, small-scale prepara-tion of highly pure total RNA from cultured cells, animal tissues and plant tissues. The kit use an unique cell lysis buffer to achieve isolation of RNA rapidly and conveniently. The proce-dure avoids the phenol and chloroform extraction. Cells or tissues are lysed by incubation in the lysis buffer after homogenization or grinding in the presence of liquid nitrogen. With the gDNA Eraser Spin Column (for remove the genome DNA) and RNA Spin Column (for binding RNA), highly pure and quality RNA can be obtained.The protocol provides a simple method to achieve the rapid isolation of highly pure RNA and the entire procedure can be accomplished within 20 minutes after tissues or cells being lysed. The pure RNA obtained by the kit basically contain no protein and have no genomic DNA contamination. The protocol allows the purification of about 10 - 30 mg of high quality of RNA from 1.0 x 105 - 1.0 x 107 cultured cells , 5 - 40mg of mammalian tissues, and 50 - 100 mg plant tissues. The highly pure RNA eluted with RNase-free H2O can be used in Northern blot, dot blot, mRNA purification, in vitro translation, RNase protection assay, RT-PCR, real time RT-PCR, cDNA library construction and other kinds of molecular experiments.Using the gDNA Eraser Spin Column can isolate easily genome DNA which can be used in PCR reaction and other kinds of molecular experiments*1.

*1 : For gDNA extraction, follow the gDNA Extraction Protocol in the Appendix.

II. Kit Components (50 reactions)

The kit contains Part I and Part II.

■ Part I50X DTT SolutionRecombinant DNase I (RNase-free；5 U/ml)10X DNase I Buffer

700 ㎕1000 U

1 ml

■ Part IIBuffer RL*2Buffer RWA*2

Buffer RWB*3

RNase Free dH2OgDNA Eraser Spin ColumnRNA Spin ColumnCollection Tube (2 ml)RNase Free Collection tube (1.5 ml)

32 ml28 ml30 ml15 ml

50505050

*2: Contain strong denaturant. Be careful to avoid contacting with the skin and eyes. In the case of such contact, wash immediately with plenty water and seek medical advice.

*3: Before the first use of the kit, add 70 ml of 100% ethanol to Buffer RWB.

[Reagents not supplied in this kit]

◆ 100% ethanol◆ 70% ethanol( prepare by 0.1% DEPC treated distilled water)◆ PBS

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III. Storage and Shipping

1. PartIshouldbeshippedandstoredat-20℃.2. PartIIcanbestoredandshippedatroomtemperature(15-25℃).

IV. Precautions for Preventing RNase Contamination

ImportantaspectsintheisolationofRNAistoinhibitRNasespresentincellsandtopreventcontaminationofRNasespresentinallinstrumentsandreagents.Therefore,thefollowingprecautionsshouldbeadopted,

-Wearcleandisposablelatexgloves.-OperatetheprotocolofisolationofRNAontheexclusiveexperimentarea.-Avoidtalkingintheoperation.andsoon.

Bytheseprecaution,RNasespresentinthesweatandsalivaoftheoperatorcanbemini-mized.Ifusedglassware,treatthemaccordingtothefollowingprotocol:

◆ Treattheglasswareat37℃for12hoursbyincubationinwatercontaining0.1%DEPC(diethylpyrocarbonate).TheninordertoremovetheresidualDEPC,autoclaveat120℃for30minutes.

◆ Preparethereagent.TheglasswaretopreparethereagentusedforRNAisolationshouldbetreatedbyhot-airsterilizationmethodortheabovemethod.DisposableplasticwarealternativelycanbeusedforRNAisolation.Thedistilledwaterusedinthereagentshouldbetreatedwith0.1%DEPCandthensterilizebyautoclave.ThespecificreagentanddistilledwaterusedforRNAisolationshouldnotbeusedmixtoavoidcrosscontamination.

V. Precautions before Starting

1. IfaprecipitateappearinBufferRL,warmitat60℃todissolvetheprecipitate.Useitaftercoolingdowntoroomtemperature.

2. Addappropriatevolumeof50XDTT(Dithiothreitol)SolutionintoBufferRLtoafinalconcentrationof2%beforeuse.Namelyadd20μlof50XDTTSolutioninto1mlofBufferRL.Itshouldbepreparedimmediatelybeforeuse.Themixturecanbestoredatroomtemperaturefor1month.

3. Beforethefirstuseofthekit,add70mlof100%ethanoltoBufferRWB,andmixwell.4. ThemaximalloadingcapacityofgDNAEraserSpinColumnandRNASpinColumnare

each600μl.Iflargervolumeistobeprocessed,loadthesampletothecolumnintoseveraltimes.

5. Allstepsoftheprotocolshouldbecarriedoutatroomtemperatureifthereisnospe-cialinstruction.

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VI. Sample Amount and Buffer RL Volume

TheamountofstartingmaterialoftissueandcellshaveagreatinfluenceonRNAyieldandpurity.TheyieldandqualityofRNAwillbelowerwhenusingmorestartingmaterial.Gener-ally,RNAcanbepurifiedfrom1.0x105-1.0x107culturedcells,5-40mgoffrozenorfreshmammaliantissuesand50-100mgoffrozenorfreshplanttissues.Werecommendtousetheappropriateamountofculturedcells1.0x106,10mgforanimaltissues,and50mgforplanttissues.Itisrecommendedtoreducetheamountofstartingmaterialsifthetissues,suchasspleen,kidneyandsoon,arerichingenomicDNA.AnditisrecommendedtoreducetheamountofstartingmaterialandincreasethevolumeofBufferRLforsomeplanttissue(stemorrootandsoon)andsomehardmammaliantissue(lung,heartandsoon).Foropti-malrecovery,refertoTable1fortheamountofstartingmaterialsofdifferenttissueandthevolumeoflysisBufferRL.

SampleAmount RecommendedvolumeofBufferRL

Adherentcells(lessthan6cmdiameterdish) 350μlAdherentcells(6-10cmdiameterdish) 600μlLessthan5x106ofsuspensioncells 350μl5x106-107ofsuspensioncells 600μl5-20mgofordinaryanimaltissue(e.g.,brain,liver) 350μl20-40mgofordinaryanimaltissue(e.g.,brain,liver) 600μl5-20mgofspecialtissue(e.g.,lung,kidney,spleen) 350μl20-40mgofspecialtissue(e.g.,lung,kidney,spleen) 600μl50-100mgofplanttissue(leaf,stem) 500μl

Table1.StartingamountofdifferentsamplesandBufferRLvolume

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VII. Protocols

●Flow chart

Materials

BufferRWA

BufferRWB

GrindinLiquidnitrogenorhomogenizeAddBufferRL

TransferlysatetogDNAEraserSpinColumn

Addequalvolumeof70%ethanoltotheflow-through

LoadthemixturetoRNASpinColumnforbindingRNA

｝WashRNASpinColumnColumn

DNaseIdigest(optional)

BufferRWBwash

AddRNaseFreedH2OtoeluteRNA

RNASolution

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Forculturedmammaliancells

1.Celllysis:● Lysisofsuspensionculturedmammaliancells

(1) Centrifugecellcultureofsuspensioncellsat8,000Xgat4℃for2minutes,thendiscardthesupernatant.

(2) Resuspendthecellpelletwith1XPBS,andcentrifugeat8,000Xgat4℃for2min-utes,discardthesupernatant.

(3) Addappropriatevolume(referTable1)ofBufferRL(makesurethat50XDTTSolu-tionhasbeenaddedtoBufferRL)tothecellpellets.

(4) Pipetupanddownuntilthelysatehasnoprecipitate.

● Lysisofadherentcells(1) Discardtheculturemediumthenwashthecellwith1XPBS.(2) Addappropriatevolume(referTable1)ofBufferRL(makesurethat50XDTTSolu-

tionhasbeenaddedtoBufferRL)totheadherentcells.Spreadthebufferoverthesurfaceofthecellsthenincubateatroomtemperatureforamomentinordertolysethecells.Scrapeoffthecellsbypipetting(forcellstightlyadherenttothecultureflask,acellscrapercanbeusedtoscrapeoffthecells).

(3) Transferthecelllysatetoacentrifugetube,pipetupanddownuntilthelysatehasnoprecipitate.

2. Incubatethecelllysateatroomtemperaturefor2minutes.3. SetgDNAEraserSpinColumnina2mlCollectionTube(suppliedbythekit).4. ApplythelysatetogDNAEraserSpinColumninCollectionTube(2ml).5. Centrifugeat12,000rpmfor1minutes.6. DiscardgDNAEraserSpinColumn(donotdiscardwhenyouisolategenomicDNA.See

Appendix).Addequalvolumeof70%ethanoltotheflow-throughinthe2mlCollectionTube(aprecipitatemayappear)andmixthoroughlybypipettingupanddown.

7. Applythemixture(includinganyprecipitate)toRNASpinColumnin2mlCollectionTube.(Ifthevolumeofthemixtureismorethan600μl,themixtureisappliedbydividingintoseveraltimes.Neverloadthemixtureover600μlateachtime.)

8. Centrifugeat12,000rpmfor1minute.Discardtheflow-through.PlacetheRNASpinCol-umnbackintothe2mlCollectionTube.

9. Add500μlBufferRWAtoRNASpinColumn.Centrifugeat12,000rpmfor30seconds.Discardtheflow-through.

10.Add600μlBufferRWBtoRNASpinColum.Centrifugeat12,000rpmfor30seconds.Discardtheflow-through.Note:Makesurethattheindicated100%ethanolhasbeenaddedtoBufferRWB. AddBufferRWBalongthewalloftheRNASpinColumn,itisbettertocompletely

removetheresidualsaltonthewall.11.DNaseIDigest(optional) BythegDNAEraserSpinColumnandRNASpinColumn,themostgenomicDNAinthe

culturedcellscanbeeffectivelyremoved.Ifthedownstreamexperimenthasastrictlyde-mandonthepurityofRNA,genomicDNAdigestioncanbecarriedoutbyaddingDNaseItothesilicamembraneoftheColumn.(1) PreparetheDNaseImixture:ina1.5mlmicrotube,add5μl10XDNaseIBuffer,4μl

RecombinantDNaseI(RNase-free,5U/μl）to41μlRNasefreedH2O.Mixgentlybyinvertingthetube.

(2) Apply50μltheDNaseImixturedirectlyontothecenterofthesilicamembraneoftheRNASpinColumn.Incubateatroomtemperaturefor15minutes.

(3) Add350μlRWBtotheRNASpinColumn.Centrifugeat12,000rpmfor30seconds.Discardtheflow-through.

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12.Repeatstep10.13.SettheRNASpinColumnbackinthe2mlCollectionTube.Centrifugeat12,000rpmfor2

minutes.14.PlacetheRNASpinColumninanew1.5mlRNaseFreeCollectionTube(supplied).Add50

-200μlRNase-freewaterdirectlytotheRNASpinColumnmembrane.Incubateatroomtemperaturefor5minutes.

15.Centrifugeat12,000rpmfor2minutestoelutetheRNA.16.IftheyieldoftheRNAislow,useanother50-200μlRNase-freewatertotheSpinCol-

umntoincreasetheyield.IfhighRNAconcentrationisrequired,applytheelutionfromstep15backtotheRNASpinColumnagain.Incubateatroomtemperaturefor5minutes.Centrifugeat12,000rpmfor2minutestoelutetheRNA.

Formammaliantissues

1. Tissuelysis● Homogenizationofmammaliantissuesaccordingtooneofthefollowing3methods.

(1)Weightthefreshorfrozenmammaliantissueandplaceitintoamortarpre-cooledwithliquidnitrogen.Freezeimmediatelythetissueinasmallvolumeofliquidnitrogen.Grindthetissuetofinepowderinthecontinuouspresenceofliquidni-trogen.TransferthepowdertissueintoaRNase-free1.5mlmicrotubepre-cooledwithliquidnitrogen.Addappropriatevolume(seeTable1)ofBufferRL(makesurethat50XDTTSolutionhasbeenaddedtotheBufferRL).Pipetupanddownuntilthelysatehasnoprecipitate.

(2) Placetheweighed(freshorfrozen)tissuesintoaRNase-free1.5mlmicrotube.Addappropriatevolume(seeTable1)ofBufferRL(makesurethat50XDTTSolutionhasbeenaddedtotheBufferRL).Homogenizeimmediatelythetissuebyvigorousagitationinthepresenceofbeadsandlysisbufferusingatissuehomogeneousdevice.Pipetupanddownuntilthelysatehasnoprecipitate.

(3) Placetheweighed(freshorfrozen)tissueinasuitablesizedvessel.Addappropri-atevolume(seeTable1)ofBufferRL(makesurethat50XDTTSolutionhasbeenaddedtotheBufferRL).Homogenizeimmediatelythetissueusingarotor-statorhomogenizerontheiceuntilitisuniformlyhomogeneous.TransferthelysatetoaRNase-free1.5mlmicrotube.

● HomogenizationofthemammaliantissuesfordifficultdisruptionorrichingenomicDNA.Weighafreshorfrozenmammaliantissueandplaceitintoamortarpre-cooledwithliquidnitrogen.Freezequicklyinasmallvolumeofliquidnitrogen.Grindthetissuetofinepowderinthecontinuouspresenceofliquidnitrogen.TransferthepowdertissueintoRNase-free1.5mlmicrotubepre-cooledliquidnitrogen.Addappropriatevolume(seeTable1)ofBufferRL(makesurethat50XDTTSolutionhasbeenaddedtotheBuf-ferRL).Pipetupanddownuntilthelysatehasnoprecipitate.Note:Completedisruptionandhomogenizationofthestartingmaterialisakeyre-quirementforobtainingofhighlyqualitytotalRNA.Ifthelysateisviscosity,itcanpassthrougha20-gaugeneedleattachedtoasterileplasticsyringeatleast5-10timestoclearthelysate.

2. Centrifugeat12,000rpmfor5minutesat4℃3. SetagDNAEraserSpinColumnina2mlCollectionTube(suppliedinthekit).4. ApplythelysatetothegDNAEraserSpinColumninthe2mlCollectionTube.5. Centrifugeat12,000rpmfor1minutes.

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6. DiscardgDNAEraserSpinColumn(donotdiscardwhenyouisolategenomicDNA.SeeAppendix).Addequalvolumeof70%ethanoltotheflow-throughintheCollectionTube(theremayappearprecipitate)andmixthoroughlybypipettingupanddown.

7. Applythemixture(includinganyprecipitate)toRNASpinColumnina2mlCollectionTube.(Ifthevolumeofthemixtureismorethan600μl,repeattheprocedure.Butdonotloadthemixturemorethan600μl.)

8. Centrifugeat12,000rpmfor1minute.Discardtheflow-through.SettheRNASpinCol-umnbackintothe2mlCollectionTube.


10.Add600μlBufferRWBtoRNASpinColum.Centrifugeat12,000rpmfor30seconds.Dis-cardtheflow-through.Note : Makesurethattheindicated100%ethanolhasbeenaddedtoBufferRWB. AddBufferRWBalongthewalloftheRNASpinColumn,itisbettertocompletely

removetheresidualsaltonthewall.11.DNaseIDigest(optional) BythegDNAEraserSpinColumnandRNASpinColumn,mostgenomicDNAinthetis-

suescanbeeffectivelyremoved.IfthedownstreamexperimenthasastrictlydemandonpurityofRNA,genomicDNAdigestioncanbecarriedoutbyaddingDNaseItothesilicamembraneoftheColumn.(1) PrepareDNaseImixture:Ina1.5mlmicrotube,add5μl10XDNaseIBuffer，4μl

RecombinantDNaseI(RNase-free，5U/μl）to41μlRNasefreedH2O.Mixgentlybyinvertingthetube.

(2) Pipet50μlDNaseImixturedirectlyontothecenterofthesilicamembraneofthecolumn.Incubateatroomtemperaturefor15minutes.


12.Repeatstep10.13.SettheColumnbackintothe2mlCollectionTube.Centrifugeat12,000rpmfor2minutes.14.PlacetheRNASpinColumninanew1.5mlCollectionTube(supplied).Add50-200μl

RNase-freewaterdirectlytotheSpinColumnmembrane.Incubateatroomtemperaturefor5minutes.

15.Centrifugeat12,000rpmfor2minutestoelutetheRNA.16.IfyieldoftheRNAislow,useanother50-200μlRNase-freewatertotheSpinColumn

toincreasetheyield.IfhighRNAconcentrationisrequired,addtheelutionfromStep15backtotheSpinColumn.Incubateatroomtemperaturefor5minutes.Centrifugeat12,000rpmfor2minutestoelutetheRNA.

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Forplanttissues1. Weightafreshorfrozenplanttissueandplaceitintoamortarpre-cooledwithliquid

nitrogen.Freezepromptlyinasmallvolumeofliquidnitrogen.Grindthetissuetofinepowderinthecontinuouspresenceofliquidnitrogen.TransferthepowdertissueintoaRNase-free1.5mlmicrotubepre-cooledwithliquidnitrogen.Addappropriatevolume(seeTable1)ofBufferRL(makesurethat50XDTTSolutionhasbeenaddedtotheBufferRL).Pipetupanddownuntilthelysatehasnoprecipitate.Note : Completedisruptionandhomogenizationofthestartingmaterialisakeyrequire-

mentforobtainingofhighlyqualitytotalRNA.Ifthelysateisviscosity,itcanpassthrougha20-gaugeneedleattachedtoasterileplasticsyringeatleast5-10timestoclearthelysate.

2. Centrifugeat12,000rpmfor5minutesat4℃.3. SetagDNAEraserSpinColumnina2mlCollectionTube(supplied).4. TransferthelysatetothegDNAEraserSpinColumnin2mlCollectionTube.5. Centrifugeat12,000rpmfor1minute.6. DiscardthegDNAEraserSpinColumn(donotdiscardwhenyouisolategenomicDNA.

SeeAppendix).Addequalvolumeof70%ethanoltotheflow-throughinthe2mlCollec-tionTube(theremayappearprecipitate)andmixthoroughlybypipettingupanddown.

7. Applythemixture(includinganyprecipitate)toaRNASpinColumnin2mlCollectionTube.(Ifthevolumeofthemixturemorethan600μl,repeattheprocedure.Butdonotloadthemixturemorethan600μl.)

8. Centrifugeat12,000rpmfor1minute.Discardtheflow-through.PlacetheRNASpinCol-umnbackintothe2mlCollectionTube.


10.Add600μlBufferRWBtoRNASpinColum.Centrifugeat12,000rpmfor30seconds.Discardtheflow-through.Note : Makesurethattheindicated100%ethanolhasbeenaddedtoBufferRWB. AddBufferRWBalongthewalloftheRNASpinColumn,itisbettertocompletely

removetheresidualsaltonthewall.11.DNaseIDigest(optional) BythegDNAEraserSpinColumnandRNASpinColumn,themostgenomicDNAinatis-

suecanbeeffectivelyremoved.IfthedownstreamexperimenthasastrictlydemandonthepurityofRNA,genomicDNAdigestioncanbecarriedoutbyaddingDNaseItothesilicamembraneoftheColumn.(1) PreparetheDNaseImixture:Ina1.5mlmicrotube,add5μl10XDNaseIBuffer,4μl

RecombinantDNaseI(RNase-free,5U/μl）to41μlRNasefreedH2O.Mixgentlybyinvertingthetube.

(2) Pipet50μlDNaseImixturedirectlyontothecenterofthesilicamembraneoftheColumn.Incubateatroomtemperaturefor15minutes.


12.Repeatstep10.13.SetRNASpinColumnbackintothe2mlCollectionTube.Centrifugeat12,000rpmfor2

minutes.14.PlacetheRNASpinColumninanew1.5mlCollectionTube(supplied).Add50-200μl

RNase-freewaterdirectlytotheSpinColumnmembrane.Incubateatroomtemperaturefor5minutes.

15.Centrifugeat12,000rpmfor2minutestoelutetheRNA.16.IfyieldoftheRNAislow,useanother50-200μlRNase-freewatertotheSpinColumn

toincreasetheyield.IfhighRNAconcentrationisrequired,addtheelutionfromStep15backtotheSpinColumn.Incubateatroomtemperaturefor5minutes.Centrifugeat12,000rpmfor2minutestoelutetheRNA.

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VIII. Experimental Examples

1. PurificationoftotalRNAfromculturedcells 10μgtotalRNAcanbeobtainedfrom1.0x106HL60cellsusingthekit(Fig.1).

M 1

M：DL2,000DNAMarker1：HL60cellRNA

Fig.1TotalRNAofculturedcells

2. PurificationoftotalRNAfrommammaliantissues HighlypuretotalRNAcanbeobtainedfrom5-10mgofvariousmousetissuesusingthe

kit(Fig.2).

M 1 2 3 4 5 6 7 8 MM:DL2,000DNAMarker1:MouseLiverRNA2:MouseHeartRNA3:MouseKidney4:MouseLungRNA5:MousePancreasRNA6:MouseSpleenRNA7:MouseBrainRNA8:MouseThymusRNA

Fig.2TotalRNAofmammaliantissues

3. PurificationoftotalRNAfromplanttissues HighlypureRNAcanbeobtainedfrom50mgleavesofmaizeandwillow(Fig.3).

M 1 2

M:DL2,000DNAMarker1:LeafofmaizeRNA2:LeafofwillowRNA

Fig.3TotalRNAofplanttissues

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IX. Yields of Total RNA from Various Materials

Samples RNAyield

Animaltissues

MouseLiver 30-50μg/10mgMouseHeart 5-10μg/10mgMouseKidney 20-30μg/10mgMousePancreas 5-15μg/10mgMouseSpleen 20-30μg/10mgMouseThymus 10-20μg/10mgMouseLung 10-20μg/10mgMouseBrain 5-10μg/10mgRatMuscle 2-4μg/10mg

PlantTissues

LeafofMaize 30-40μg/100mgLeafofgourd 10-15μg/100mgLeafofwillow 40-50μg/100mgLeafofmungbean 15-20μg/100mg

CulturedCell HL60cell 8-15μg/106cells

X. Appendix：Genomic DNA Extraction

GenomicDNAcanbesimplyextractedfromgDNAEraserSpinColumninaccordingtothefollowingprotocol.

1. LysethesamplefollowingSteps1to4intheVII.Protocols.2. SetthegDNAEraserSpinColumnbackinto2mlCollectionTubeagain.3. Add500μlBufferRWAtothegDNAEraserSpinColumn.Centrifugeat12,000rpmfor

30seconds.Discardtheflow-through.4. Add600μlBufferRWBtogDNAEraserSpinColumn.Centrifugeat12,000rpmfor30

seconds.Discardtheflow-through.Note : Makesurethattheindicated100%ethanolhasbeenaddedtoBufferRWB. AddBufferRWBalongthewallofthegDNAEraserSpinColumn,itisbetterto

completelyremovetheresidualsaltonthewall.5. RepeatStep4.6. PlacethegDNAEraserSpinColumnbackintothe2mlCollectionTube.Centrifugeat

12,000rpmfor2minutes.7. PlacethegDNAEraserSpinColumninanew1.5mlRNase-freeCollectionTube(sup-

plied).Add50μldH2OorTEBufferandincubateatroomtemperaturefor5minutes.8. Centrifugeat12,000rpmfor2minutestoelutethegenomicDNA.

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XI. Troubleshooting

Q1: WhyisaRNASpinColumnclogged?A1: Thereasonsarealmostthefollowing.

(1) Incompletelysisofsamples.TheincompletelysisofthesamplemayresultintheblockofRNASpinColumn.Seedetailsondisruptionandhomogenizationmethodoftissueorcells.Itisrecommendedtousethemethodofgrindingsamplewithapestleandmortarinthepresenceofliquidnitrogentodisrupttissuesamples.

(2) Toomuchstaringmaterialshavebeenused.Toomuchsampleshavemorequan-tityofnucleicacidwhichcanclogtheColumn.See“VI.SampleamountandBufferRLvolume”fordetailsontheamountofstartingmaterialandthevolumeofBufferRL.

(3) Centrifugationtemperatureshouldbeat20-25℃.Toolowortoohightempera-turewouldaffectperformanceofRNASpinColumnandcloggedtheColumn.

Q2: WhyistheyieldoftotalRNAlower?A2: (1) Insufficientdisruptionandhomogenization.Itisessentialtolysethesamplecom-

pletely.Seedetailsondisruptionandhomogenizationmethodoftissueorcellsinprotocols.

(2) Toomuchstaringmaterial.See“VI.SampleAmountandBufferRLVolume”forde-tailsontheamountofstartingmaterialandthevolumeofBufferRL.

(3) EluteRNAinsufficient.WerecommendeluteRNAonceagain.Incubationtimecanprolongupto10minutesafteraddRNase-FreedH2OtotheSpinColumn.

(4) Elutioncontainsresidualethanol.DuringthesecondwashwithBufferRWB,besuretocentrifugeat12,000rpmfor1minutetodryRNASpinColumnmembrane.Thenwerecommendtoperformanother2minutescentrifugationstepasdescribedintheprotocols.ResidualethanolwilloccurdecreaseofRNAyield.

Q3: WhyispurifiedRNAdegraded?A3: (1) Samplesarenotfresh.Usefreshsamplesasmuchaspossibleorfreezethesamples

inliquidnitrogenandthenstoreat-80℃.(2) RNaseispresentinthereagentsandmaterials.Beforestartingtheprocedure,see“IV.

PrecautionsforPreventingRNaseContamination”thoroughly.(3) ThesampleisrichinRNase.Itisrecommendedtouselessstartingmaterialand

increasethevolumeofBufferBLfortissuesandcellsrichinRNase.

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Q4: WhydoesthepurifiedRNAhavegenomicDNAcontamination?A4: BythegDNAEraserSpinColumn,themostgenomicDNAinthesamplecanbeeffec-

tivelyremoved.Generally,thepurifiedRNAdoesnotcontaingenomicDNA.Ifcontami-nationwithgenomicDNAinRNAoccurs,thereasonsaremainlythefollowing.(1) Insufficientdisruptionandhomogenization.Itisessentialtolysethesamplecom-

pletely.ItwouldresultintheresidueofgenomicDNA.Seedetailsondisruptionandhomogenizationmethodoftissueandcellsintheprotocols.

(2) Toomuchstaringmaterialused.Moreamountofsamplecanproducemorequan-tityofnucleicacidthatwouldexceedtheloadingcapacityofgDNAEraserSpinCol-umn,andresultintheresidueofgenomicDNA.See“VI.SampleAmountandBufferRLVolume”fordetailsontheamountofstartingmaterialandthevolumeofBufferRL.

(3) SamplesrichingenomicDNA.GenomicDNAishighinsomematerials.Itisrec-ommendedtouselessstartingmaterialandincreasethevolumeofBufferBLforsamplerichingenomicDNA.

(4) DNaseItreatmentisomitted.Ifthedownstreamexperimenthasastrictlyrequir-mentonthepurityofRNA,orasampleisrichingenomicDNA,DNaseIdigestionshouldbecarriedoutaccordingtotheDNasedigestionstepintheprotocols.

Q5: WhatisameaningoftheabsorbancevaluesofRNA?A5:Thevaluesof260nm,320nm,and280nmrepresenttheabsorbanceofnucleicacid,

background(turbidityofreagent),concentrationofsaltandprotein,respectively.ThevalueofOD260/280(R)representthedegreeofcontaminationoforganicssuchaspro-teinandsoon.Thevalueshouldbebetween1.8and2.2forhighlyqualityRNA,theratiolowerthan1.8meansthatthecontaminationofproteinisobviousandhigherthan2.2meansthattheRNAisdegradedtosinglenucleicacid.DilutetheRNAusingTEbufferwhenchecktheRNAabsorbancevalues.

Q6: HowtocalculatetheconcentrationofRNA?A6: CalculatetheconcentrationofRNAaccordingtheabsorbanceofRNAatdifferentwave-

length: RNA(μg/μl)=（OD260-OD320）XdilutionX0.04μg/μl

Q7: HowintegrityisRNAextracted?A7: UsingTaKaRaMiniBESTUniversalRNAExtractionKit,RNAlongerthan200basescanbe

efficientlypurified.

Q8: HowqualityisthegenomicDNAextracted?A8: ThegenomicDNAextractedbythiskitisbasedonanoptionalprotocolandtheyieldof

genomicDNAislowerthanothergenomicDNAextractionkit.TheobtainedgenomicDNAisonlyusedbyPCRastemplate.ForextractionofgenomicDNA,useTaKaRaMini-BESTUniversalGenomicDNAExtractionKit5.0（Cat.#9765）inthecasethatlowcontentofgenomicDNAinsamplesandthatrequirementofhigherpurityofgenomicDNAindownstreamexperiments.

Allmarksarethepropertyoftheirrespectiveowners.Certaintrademarksmaynotberegisteredinalljurisdictions.

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16 URL:http://www.takara-bio.com

NOTE : Thisproductisforresearchuseonly.Itisnotintendedforuseintherapeuticordiagnosticproceduresforhumansoranimals.Also,donotusethisproductasfood,cosmetic,orhouseholditem,etc.Takaraproductsmaynotberesoldortransferred,modifiedforresaleortransfer,orusedtomanufacturecommercialproductswithoutwrittenapprovalfromTAKARABIOINC.Ifyourequirelicensesforotheruse,pleasecontactusbyphoneat+81775437247orfromourwebsiteatwww.takara-bio.com.Youruseofthisproductisalsosubjecttocompliancewithanyapplicablelicensingrequirementsdescribedontheproductwebpage.Itisyourresponsibilitytoreview,understandandadheretoanyrestrictionsimposedbysuchstatements.Alltrademarksarethepropertyoftheirrespectiveowners.Certaintrademarksmaynotberegisteredinalljurisdictions.

TaKaRa MiniBEST Universal RNA Extraction KitCat.#9767v201306Da

Documents

5B,B3B.JOJ#&45 6OJWFSTBM3/&YUSBDUJPO,JU … · 4 TaKaRa MiniBEST Universal RNA Extraction Kit Cat. v00Da RL:http: III. Storage and Shipping 1. Part I …