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如何利用ligationmediated pcr 检测插入了什么基因
ThisprocedurewasfirstdescribedbyBertrandetal(ProceedingsoftheNationalAcademyofScienceUSA(1993)Vol.90pp.3496-3500,andNucleicAcidsResearch(1994)Vol.22pp.293-300)todemonstratethatribozymescouldbeenzymaticallyactiveinvivo.WeadaptedthemethodtoshowthatcertainoligodeoxynucleotidescoulddirecttheactivityofendogenousribonucleaseHtocleavetargetmRNAinintacthumanleukaemiacellsfollowingstreptolysinO-mediateddelivery(method)oftheantisenseeffectors(reference1,reference2).ThetheoryofreverseligationmediatedRT-PCR(RL-PCR)isdescribedelsewhereinthesepages.
RL-PCRcanbebrokendownintoanumberofsimplesteps.SynthesisbyinvitrotranscriptionandpurificationofanRNAlinkerspecies.ExtractionoftotalRNAfromcellsintowhicholigodeoxynucleotidehasbeendeliveredby,forexample,streptolysinOpermeABIlization.LigationoftheRNAlinkertoallavailable5"monophosphatesinthepurifiedtotalcellularRNAsample.ReverseTranscriptionoftheRNAusingagene-specificprimer.AmplificationofspecificfragmentsbyPCRusinglinker-specificandgene-specificprimers.Sub-amplificationofthefirstPCRproductusinglinkerspecificandnested,labelled,genespecificprimers.AlternativelythefirstPCRmaybesequencedusingthenested,labelled,gene-specificprimer.
Reagents/Solutions
- 5µMTemplateforlinkerRNAtranscript(duplexofRLPCRoligonucleotides1and2)in30mMNaCl,2mMTris-HCl,0.2mMEDTApH8.0.
- 5xTranscriptionBuffer.
- 0.2MTris-HClpH7.5
- 50mMNaCl
- 10mMspermidine
- 30mMMgCl2
- 0.1Mdithiothreitol
- 1MMgCl2
- BacteriophageT7RNApolymerase(200U/µl)
- RNaseInhibitor(~40U/µl)
- 100mMNTPs
- 20%Acrylamide-bis(19:1),1xTBEbuffer.
- 4Mguanidinethiocyanate,5mMsodiumcitratepH7,0.5%sarkosyl
- To50gGuanidinethiocyanateadd
- 58.6mlH2O
- 3.52ml0.75MsodiumcitratepH7.0
- 5.28ml10%sarkosyl
- 2MsodiumacetatepH4
- 2-mercaptoethanol
- watersaturatedphenolpH4.0-4.3
- Chloroform:isoamylalcohol(49:1byvolume)
- Isopropanol
- T4RNAligase(6U/µl)+10xreactionbuffer(Boehringer).
- RNaseInhibitor(RNaseBlockI40U/µl,Stratagene).
- DNaseI(20-30U/µl)(Boehringer).
- Linearpolyacrylamide(2.5µg/µl,Gaillard&Strauss,NucleicAcidsResearch1990Vol.18pp.378).
- 10mMEDTApH8.0.
- Pfupolymerase(native)+10xreactionbuffer1(Stratagene).
- SuperscriptRTase(Gibco).
- Gene-Specificreversetranscription(RT)primer.
- dNTPs10mMeach.
- RNaseA1ng/µl(boiled)
- RNALinkerspecificprimer(RLPCRoligonucleotide3).
- Nestedgenespecificprimer(GSP).
- Labelled,nested,secondgenespecificprimer(DigP).
- SequencinggelloADIngbuffer(95%formamide,10mMNaOH,0.05%bromophenolblue,0.05%xylenecyanol)
- Sequencinggelmix(6-8%polyacrylamide:bis(19:1),7Murea,1xTBEbuffer)
- SequencingGradeTaq+5xreactionbuffer(Promega).
- d/ddNTPmixes(20µMdATP,dCTP,dTTP,7-deazadGTP:Amix350µMddATP,Cmix200µMddCTP,Gmix30µMddGTP,Tmix600µMddTTP).
- Ethanol,icecold.
- Lightmineraloil.
TranscriptionandpurificationofRNAlinker
- HybridizeRLPCR1(5"...TTTCAGCGAGGGTCAGCCTATGCCCTATAGTGAGTCGTATTA...3")andRLPCR2(5"...TAATACGACTCACTATAG...3").
- Add100µl100µMRLPCR1to100µl100µMRLPCR2and50µl1xTEN(150mMNaCl,10mMTris-HCl,1mMEDTApH8.0)
- Heatto70°Cfor5minutes,allowtocooltoroomtemperatureslowly.
- Analyseasmallaliquotbynon-denaturingpolyacrylamidegelelectrophoresistoensurethathybridhasbeenformed.
- Dilutehybridto5µMbyadditionof1750µl0.2xTEN
- Transcriptionreaction.(ThesearetheconditionsofMilliganetalNucleicAcidsResearch1987Vol.15pp.8783-8798).PlacethefollowinginanEppendorfinthefollowingorder.
- 153µlH2O
- 50µl0.1Mdithiothreitol
- 7µl1MMgCl2
- 100µl5xtranscriptionbuffer
- 20µl100µMATP
- 20µl100µMCTP
- 20µl100µMGTP
- 20µl100µMUTP
- 20µl5µMRLPCR1/RLPCR2hybrid
- 20µlRNaseinhibitor(~40U/µl)
- 50µlT7RNApolymerase(200U/µl)
- Incubatetranscriptionreactionfor6hoursat37°C.
- Fractionatereactiontemplateandproductsbyelectrophoresisthrougha20%non-denaturingpolyacrylamidegel.
- IdentifythetranscriptbyUV-shaddowingoverathinlayerchromotographyplate.Cutoutthebands.
- Mashthegelsliceswhichcontainthelinkertranscript.
- ExtractthelinkerRNAfromthegelbyconstantagitationin~2volumesoflysisbuffer(20ml4MGuCSNbuffer+2ml2MsodiumacetatepH4.0+200µl2-mercaptoethanol)overnight.Centrifuge(10-15,000g,10minutes)topelletthemashedgel.Collectthesupernatent.
- Re-extractthemashedgelsliceswiththesamevolumeoflysisbufferasabove,butfor2hours.
- PoolthecollectedsupernatentandprecipitatetheRNAbyadditionof1volumeofisopropanolfollowedbyincubationat-20°Covernight.
- PellettheprecipitatedRNAlinkerbycentrifugationat10-15,000gfor30minutes.
- ResUSPendtheRNAin550µllysisbufferandtreatasifpurifiyingtotalRNAfromcells.
- ResuspendtheresultantRNApelletin100µlH2Oanddivideinto10x10µlaliquots.
- Storeat-20°Cuntilrequired.
LigationoflinkerRNAtototalcellularRNA
- Toeachreactiontubeadd1µgoftotalcellularRNAin4µlddH2O.Storeonice.
- Makemastermixcontainingthefollowingperreaction:
- 1µl10xligasebuffer,
- 1-2µlgelpurified25-merRNA(~500ng)
- 1µlRNaseinhibitor
- 0.5µlT4RNAligase(6U/µl)
- 0.5µlDNaseI(~25U/µl)
- adjustvolumeto6µl/reactionwithH2O(1-2µl)
- Add6µlofmastermixtoeachreactiontube.
- Incubateovernightat17°C.
- Incubateat37°Cfor30minutes(ensures~completeDNaseIdigestion).
- Stopreactionbyadditionof5µl10mMEDTApH8.0and200µlofmodifiedlysisbuffer(lysisbuffer:phenolpH4.31:1byvolume),whichcontains5µglinearpolyacrylamide(co-precipitant)per200µl.Vortexbriefly.
- Inducephaseseparationbyadditionof25µlchloroform.
- Recovertheupperaqueusphasetoathermalcyclertubeandprecipitatenucleicacidswith1volume(ca.130µl)propan-2-ol.Storeat-20°Covernight
ReverseTranscription
- Spindownprecipitatesat10-15,000gfor30minutes.Removeanddiscardsupernatant.Briefsecondspin,pulsetofullspeedthenrelease,discardthe2-4µlofsupernatantsocollected.
- Resuspendtheprecipitatesin4µlH2O.Storeoniceuntilreadytoproceed.
- MakeRTmastermix1withthefollowingcomponentsandwiththisvolumeperreaction
- 0.9µlH2O
- 0.7µlPfupolymerasebuffer1
- 0.7µl0.1Mdithiothreitol
- 0.2µl10µMgene-specificRTprimer
- 0.5µlRNaseBlockI
- HeatRNAsolutionsto95°Cfor5minutesthenplaceimmediatelyat37°C.Incubatefor2-3minutes.
- Add3µlofRTmastermix1perreaction.
- Incubateat37°Cfor45minutes.
- MakeRTmastermix2withthefollowingvolumesperreaction.
- 1.4µlH2O
- 0.3µlPfupolymerasebuffer1
- 0.3µl0.1Mdithiothreitol
- 0.5µl10mMdNTPs(each)
- 0.5µlSuperscriptRTase
- Add3µlRTmastermix2perreaction.
- Incubateat37°Cfor45minutes.
- Incubateat95°Cfor10minutes.
- Add1µlRNaseA.
- Incubateat37°Cfor20minutes.
- StoreoniceuntilreadyforPCR1.
FirstPCR
- MakePCR1mastermixwiththefollowingvolumesperreaction.
- 4.8µlH2O
- 1µlPfupolymerasebuffer1
- 2µl10µMRLPCR3(linkerspecificprimer,5"...GGGCATAGGCTGACCCTCGCTGAAA...3")
- 2µl10µMGSP
- 0.2µlPfupolymerase(~0.5U)
- Add10µlPCR1mastermixtoeachRTreaction.
- Overlaywith50µllightmineraloil.
- Thermalcycle:~20cycles.
SecondPCR
- MakePCR2mastermixwiththefollowingvolumesperreaction.
- 9.5µlH2O
- 1.5µlPfupolymerasebuffer1
- 1.5µl10µMRLPCR3
- 2µl10µMlabelledprimer
- 0.3µl10mMdNTPs
- 0.2µlPfupolymerase
- Place15µlPCR2mastermixintofreshthermalcyclertubes.
- Overlaywith50µllightmineraloil.
- Add5µlPCR1reaction.
- Thermalcycle:~5to10cycles.
- Remove2µlto8µlsequencinggelloadingBuffer.
- Heatto95°Cfor10minutes.
- Quenchonice,storeoniceuntilanalysed.
- Fractionatethrough6-8%polyacrylamidesequencinggel(geltemperature50°C).
- Standarddetectionprocedureforyourlabeltype.Digoxigeninorfluoresceinlabelledprimerswillrequirethenucleicacidsinthegeltobeelectroblottedordiffusionblottedontonylonmembranepriortochromogenicdetection(method).
RL-PCRSequencing
Pleasenotethisprotocolisanadaptionofthe(Promega)fmolesequencingprocedure.
- Remove2µlofPCR1to18µlH2O.
- Place2µlofthed/ddmixesintoeachoffourtubes(iean"A"tube,a"C"tubeetc.)
- Sequencingmix:
- 8µlH2O
- 5µl5xsequencingreactionbuffer
- 2µl1µMlabelledprimer
- 1µlofthedilutedPCR1reaction
- 5UPromegasequencinggradeTaqpolymerase
- Add4µlofsequencingmixtod/ddNTPtubes.
- Overlaywith25µllightmineraloil.
- Thermalcycle:~30cycles.
- Add4µlsequencinggelloadbuffer,vortexandcentrifugetomix.Incubateat90°Cfor>5minutes.
- Loadhotsamplesontostandardsequencinggelwhichhasbeenpre-heatedto50°C.
- Fractionatebyelectrophoresis.
- Standarddetectionprocedureforyourlabeltype.
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