Real Time PCR Operation ProcedureWord文档格式.docx
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Real Time PCR Operation ProcedureWord文档格式.docx
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2Equipment9
3Experimentprocedures9
3.1Preparethestandarddilutionseries10
3.2Dilutionofsamples10
3.3Preparethereactionmix13
3.4Preparethereactionplate13
3.5Preparetherun14
3.6DataAnalysis13
4DatamanagementandRecordsmanagement14
5QualityAssessment/QualityControl16
References17
ApplicabilityandLimit
InthisexperimentapplytotheuseStepOnePlus™RealTime-PCRamplificationoftheDNAPE,SElibraryandthetranscriptome,expressionprofiling,smallRNAlibrarypreparationgroupfortheprecisequantificationbeforeontheclusterstation.
Abstract
QuantitativePCRexperimentsbasedonAgilent2100Bioanalyzerresultsof
testsonthelibraryasareference.Themainprocedureincluded:
1.thestandarddilutionseries.2.Dilutionofsamples.3.PrepareReactionMix.4.Preparethereactionplate.5.SetfortheRun.6.Runtheexperiment.7.Dataanalysis.Allprocedureneeds3to4hours.
Abbreviations
Baseline
Intheamplificationplots,alinefittothefluorescencelevelsduringtheinitialstageofPCR,whenthereislittlechangeinfluorescencesignal.
CtValue
ThePCRcyclenumberatwhichthefluorescencemeetsthethresholdintheamplificationplot.
Threshold
Inamplificationplots,theleveloffluorescenceabovethebaselineandwithintheexponentialgrowthregion.
Materials&
Method
1.ReagentsandConsumables
1.1Reagents
ReagentsName
Lot.No.
Factory
10XExTaqBuffer
AA6901A
TaKaRa
dNTPmixture
BG4401A
MgSO₄
0010805
NEWEnglandBioLABS
ROXReferenceDye
761819
Invitrogen
PEPrimer1.1
M165842
Sangon
PEPrimer2.1
M100658
SEPrimer1.1
M76070
SEPrimer2.1
M67154
NGEXPrimer1.1
M142353
NGEXPrimer2.1
M142354
Evagreen
9E0713
Biotium
TaqmanProbe
EXTaqHS
CN6501AA
DMSO
Sangon
Betaine
Sigma
Tween20XBuffer
1.2Consumables
Consumables
Lot.No.
2.0mlmicrotubes
100118-262
Axygen
1.5mlmicrotubes
100612-041
0.6mlmicrotubes
090704-301
0.2mlPCRtubes
090420-179
MicroAmp™Optical96-WellAdhesiveFilm
4311971
AppliedBiosystems
48-wellreactionplates
96-wellreactionplates
96-wellFilm
2.Equipments
Equipments
StepOnePlus™Real-TimePCRSystem
Vortex
Qilinbeier
Minicentrifuge
10D008B
EZFUGE
-20℃Fridge
4℃Fridge
3.ExperimentProcedure
3.1PreparetheStandardDilutionSeries
StandardCurve
SourceVolume(µ
L)
DiluentVolume(µ
TotalVolume(µ
Concentration(pM)
Std1
1µ
l(10nM)
9
10
1000
Std2
2µ
l(Std1)
18
20
100
Std3
l(Std2)
Std4
l(Std3)
1
Std5
l(Std4)
0.1
Figure3-1StandardDilutionSeries
1.Labeledaseparate0.2mlPCRmicrocentrifugetubeforeachStandard:
Std1,Std2,Std3,Std4andStd5.
2.AddedtherequiredvolumeofMilli-Qwater(diluents)toeachemptytube:
Tube
VolumeofDiluenttoAdd(µ
Std1
Std2
Std3
Std4
Std5
Figure3-2VolumeofDiluent
3.VortextheStandardfor10to15seconds,thencentrifugethetubebriefly.
4.Added1µ
lofStandardtotheStd1tubebyusinganewpipettetip.
5.VortexStd1for10to15seconds,thencentrifugethetubebriefly.
6.Added2µ
lofdilution1totheStd2tubebyusinganewpipettetip.
7.VortexStd2for10to15seconds,thencentrifugethetubebriefly.
8.Repeatedthestep6and7byStd2toStd3,Std3toStd4andStd4toStd5.
9.PlacedtheStandardsonicecooleruntilthereactionplateprepared.
PreparationGuidelines/Precaution:
∙Standardsarecriticalforaccurateanalysisofrundata.
∙Anymistakesorinaccuraciesinmakingthedilutiondirectlyaffectthequalityofresults.
∙Thequalityofpipettorsandtipsandthecareusedinmeasuringandmixingdilutionsaffectaccuracy.
∙UseTEbufferorwatertodilutetheStandards.
∙Pipettingupanddownisrecommendedineachstep.
3.2DilutionofSamples
1.Labeledthesamplenameonaseparate0.6mlmicrocentrifugetubeforeachdilutedsample.
2.AddedtherequiredvolumeofMilli-Qwater(diluent)toeachemptytube:
SampleVolume(µ
Samplename
99
Figure3-3VolumeofDilutedSample
3.Added1µ
lofsampletoeachtubeandvortexeachdilutedsamplefor10to15seconds,thencentrifugethetubesbriefly.
4.PlacedtheStandardsonicecooleruntilthereactionplateprepared.
∙Sampledilutionsmaybenecessarybecausethesamplevolumeislimitedto10%ofthetotalreactionvolumeintheStepOneSoftware.Thesamplemustperformdilutionsbeforeaddingthesamplestothefinalreactionmix.
∙UseTEbufferorwatertodilutethesamples.
3.3PreparetheReactionMix
1.Labeleda2.0mlmicrotubeandcoveredanaluminumfoilforthemastermix:
PEMIX.
2.Addedtherequiredvolumesofeachreagenttothetube:
Reagents
ReactionVolume(1X)(µ
l)
ReactionVolume(50X)(µ
10XTaqBuffer
l
50µ
0.1µ
5µ
Tween20X
0.5µ
25µ
H2O
3.35µ
167.5µ
100µ
Figure3-4MasterMix
3.Mixthereactionmixgentlybypipettingupanddown,thencapthetube.
4.Centrifugethetubebrieflytoremoveairbubbles.
5.Labeleda1.5mlmicrotubefortheReactionMix:
reactionmix.
6.Addedtherequiredvolumesofeachreagenttothetube:
ReactionVolume(37X)(µ
PEMix
7.05µ
260.85µ
dNTP
0.8µ
29.6µ
Probe
0.25µ
9.25µ
ROX
0.2µ
7.4µ
Primer1.1
0.3µ
11.1µ
Primer2.1
3.7µ
Figure3-5ReactionMix
7.Placethereactionmixoniceuntilthereactionplateprepared.
∙Maskandgloveshouldbewearingbeforeexperimentand70%alcoholshouldbeusedtocleanupthebench.
∙Iftheexperimentincludesmorethanonetargetassay,preparethereactionmixforeachtargetassayseparately.
∙Keepthereactionmixprotectedfromlight.Excessiveexposuretolightmayaffectthefluorescentprobes.
∙Mixthemastermixthoroughlybyswirlingthebottle.
∙Thawanyfrozensamplesbyplacingthemonice.Whenthawed,resuspendthesamplesbyvortexing,thencentrifugethetubesbriefly.
∙Pipetupanddown3timeswhenaddingTaqenzymeintothereagents.
3.4PreparetheReactionPlate
1.Puta48-wellreactionplateora96-wellreactionplateonthesupportbase.
2.Added9µ
lofReactionMixtotheappropriatewellsinthereactionplate.
lofStandardtotheappropriatewellsinthereactionplate.(Std1,Std2andStd3havetworeplicatesandStd4andStd5haveonereplicateonly)
lofSampletotheappropriatewellsinthereactionplate.(Everysampleshavethreereplicates)
5.Added1µ
lofWatertotheappropriatewellsinthereactionplatefornegativecontrolreactions.
6.Sealedthereactionplatewithopticaladhesivefilm.
7.Centrifugethereactionplatewith4000rpmin2minstoremovetheairbubbles.
8.Confirmthattheliquidisatthebottomofeachwellofthereactionplate.Ifnot,centrifugethereactionplateagainatahigherspeedandforalongerperiodoftime.
9.Placedthereactionplateoniceinthedarkuntiltherunperformed.
∙Makesureusedtheappropriateconsumables.
∙Donotallowthebottomofthereactionplatetobecomedirty.Fluidsandothercontaminantsthatadheretothebottomofthereactionplatecancontaminatethesampleblockandcauseanabnormallyhighbackgroundsignal.
3.5PreparefortheRun
1.Double-clickStepOneSoftwareshortcut.
2.ClickAdvancedSetuptocreateanexperiment.
3.ClicktheExperimentNamefield,thenenterName,DateandExperimentMethod.
4.SelectStepOnePlus™Instrument(96Wells).
5.SelectQuantitationfortheexperimenttype.
6.SelectStandardCurveasthequantitationmethod.
7.SelectTaqMan®
ReagentsorSYBR®
GREENforthereagents.
8.SelectStandard(~2hourstocompletearun)fortherampspeed.
9.SelectgDNA(genomicDNA)forthetemplatetype.
10.ClickPlateSetupontheNavigationpane.
11.SelectDefineTargetsandSamples,thenenterTargetname.(IfTaqmanisused,ReportersetFAMandQuenchersetNFQ-MGB.IfSYBRisused,ReportersetFAMandQuenchersetNONE.)Leavethedefaultinthecolorfield.
12.SelectDefineSamplesandenterSamplesname.Leavethedefaultinthecolorfield.
13.ClickAssignTargetsandSamplesandselectwellsintheviewplatelayout.
14.ClickAssignTargetstotheselectedwells(S=Standard,U=unknownSampleandN=NegativeControl)
15.ClickAssignSamplestotheselectedwells.
16.ClickRunmethodontheNavigationpane.
17.SelectReactionVolumePerwellandenterthereactionvolume10µ
l.
18.Selectthetemperatureandtimeasfollow:
HoldingStage
50℃
2minutes
95℃
10seconds
CyclingStage
94
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