iQ 荧光定量PCR仪专用96孔板_价格厂家供应商_伯乐生命医学产品(上...
来自 : 蚂蚁淘
Preparationofsaltbuffers
ThebuffersareusedforbothHPLCpurificationandSep-Pakpurification.
Highsaltbuffer(HSB):- 1Mtriethylammoniumacetate,pH8.0
- 10%acetonitrile
Lowsaltbuffer(LSB):- 25mMtriethylammoniumacetate,pH8.0
- 10%acetonitrile(thisis40-folddilutionofHSB+10%acetonitrile)
HSB,800mL
- To500mLddH2O,addapprox.25-30mLglacialaceticacid
- Slowlyadd111.3mL(80.95g)HPLCgradetriethylamine;thereactionisexothermic;thesolutionwillbecomecloudy–waituntilitclearsbeforeaddingmore)
- Adddropwiseanadditional10-15mLaceticacidtoadjustthepHto8.0.
- Add80mLacetonitrile(HPLCgrade)
- Adjustthefinalvolumeto800mLwithddH2O.
LSB,1000mL
- To25mLHSB,add100mLHPLCgradeacetonitrile
- Raisefinalvolumeto1LwithddH2O
*Filter,andthenspargebothbuffersbeforeuse.Makesenoughforapprox.9runs&2purges.DesaltingthesampleforSep-PackPurification
Materials:
- HPLCgradeacetonitrile(asetofthreerunsconsumesaround40mL)
- 20mlddH2O,0.22mmfiltered
- 15mMTEAA(thiscanbesubstitutiedwith0.750mLHSBand49.25mLddH2O,0.22mmfiltered,toproduce50mL)
- Sep-Pakcartridges,C18(Waters#20515);1columnpercydye.Canbereusedtwice.
- 12mLsyringe(1perdye)
Pre-loadprotocol:
- WashSep-Pakwith10mLofacetonitrile.
- Washwith5mLofwater.
- Washwith10mLof15mMTEAA.
SamplePreparation,LoADIng,andElution:
- Thecolumnmaybeloadedupto150A260unitsofsample(basedontheA260readingforbothlabeledandunlabeleddNTPsinthefiltrate),lyophilizeddownto3-5mL(volumesof6.5-7.5providedreasonableyields,andvolumesofupto10mLhavebeensuccessful,butwithloweryields).
- Add1MTEAAtothesample,toaconcentrationof15mM.
- Loadthesampleontothecolumn.ThedyeshouldbevisIBLenearthetopofthecolumn.Washthecolumnwith5mLof15mMTEAA.
- Elutewith3mLofacetonitrile.Discardtheinitialcolorlessdrops,collectthecoloredeluentintoa2mLcentrifugetube,anddiscardtheremainingsolution.Thecollectedvolumewillbeapproximately1.0-2.0mL.Somecoloredmaterialwillremainonthecolumnafterelution.
Immediatelywash(pre-loadprotocol)andprocessanysubsequentlots,reusingthecolumnonlytwiceforeachlot.Ifthecolumnisallowedtositformorethan5minutes,yieldsmaydecrease5-10%ormore.Avoidreusingthecolumnmorethantwotimes(atotalofthreeuses),asyieldsmaybegintodiminish.Poolthelotstogether,checkUVabsorbance,andcalculateyield.Expectedyieldsforthisstageare70-80%foreitherCydye.
HPLCPreparation
- LyophilizetheentireSep-Pakpurifiedmaterial,keepingthedyesseparate(seeLyophilizationandRedilutionsection).ResUSPendwithasmallvolume(200-500ml)of0.2MTEAA.
- Centrifugethesamplethrougha0.45mmfilter.
- Checktheabsorbanceat260nmand,ifnecessary,dilutethesampledowntoaconcentrationthatwillnotexceedthecapacityofthecolumn(DionexNucleoPac™9´250PA-100;P/N43011(anionexchange)columnlistedbelow).Typicallya1mlinjectionloopisusedtoinjecta10mMsampleofunlabeleddNTPs+Cy-dUTP.
HPLCPurification
Columnused:DionexNucleoPac™9´250PA-100;P/N43011(anionexchange)HPLCEquipment:PerkinsElmerSeries200LCPump,PerkinsElmerUV/VisDetectorLC2951.Equilibratecolumninbuffersystem,3mL/minflowrate(generalpurgemethod)
1.1.5minlowsaltbuffer(LSB)orA1.2.2mingradientto100%highsaltbuffer(HSB)orB1.3.5minHSBorB1.4.2mingradientto60%LSBorA1.5.5min60%LSBorA2.Loadsampleintoinjectionloop.See"FinalConsiderations"onthenextpageforsamplelotsizeandotherissues.
3.Rungradient(3mL/minflow,chartrecorder0.5cm/min)
3.1.5min60%LSB,40%HSB3.2.10mingradientto80%HSB3.3.1mingradientto100%HSB3.4.10min100%HSB3.5.1mingradientto60%LSB3.6.5min60%LSBFinalPreparativeProcedures
Forapreparativerunof150nmolesCy-dUTP(note"FinalConsiderations"),therecoveredfractionsofpurifiedCy-dUTPiselutedin5mlofvolatileTEAAbufferthatcanberemovedbylyophilization.LyophilizationandRedilution
(ReturntoHPLCPreparation.)- PoolfractionsofeachCydyeintoa50mLconicaltube.Punchholesintothecapusingan18-gaugeneedle.Avoidexceedingmorethanhalfthevolumeofthetube,asthesamplesmayhaveatendencytosplatterduringdegassingortravelupthetubeduringlyophilization.
- Freezethesamplesondryiceandtransferthemtoalyophilizationchamber.Wrapthechamberwithfoiltoprotectthesamplesfromlight.
- Lyophilizethesamplestocompletion(approx.12-15hrsforHPLC-purifiedsamples,6-10hrsforSep-Pak-purifiedmaterial).HPLC-purifiedsampleswillhavetheappearanceofaresidueuntilmostoftheTEAAisremoved.
Note:Steps4&5areforHPLC-purifiedsamplesonly.
- Dilutethesamplesbackupto8-10mLwith0.22mmfilteredddH2Oandlyophilizeuntilthesamplesagainresembleresidue.Thistime,itmayrequireupto24hoursoflyophilization.
- RepeatStep4.Lyophilizationiscompletewhenthesamplehastheappearanceofafilmand/orspecksofsolidresidue.Resuspendthesampleinasmallvolumeof10mMphosphatebuffer,pH7.0.BearinmindthatsomeoftheCy-dUTPwillcoattheupperregionsofthetubewallsandmaynotbereadilyvisible.Checkabsorbanceofthesample.A560/660/A260ratiosshouldbearound17forcy3and25forcy5.
Expectedoverallyields:cy3:50-70%;cy5:45-65%.
FinalConsiderations
Currently150nmolofCy-dUTPcontainedina10mMmixtureoflabeledandunlabeleddNTPsisloadedontothecolumnlistedabove,dependingonthecontentsandconcentrationofthefiltrate;PCRflow-throughsamplestendtobecleanerandmayallowforlargerinjectionlots.Thus,theflow-throughfromPCRandRTreactionsmaybekeptseparatesothattheycanbeprocessedseparately.TheSep-Pakpurificationdesaltsthesampleandislikelytoremoveotherimpuritiespresentinthesample.HPLCpurificationcanstillbeperformedwithoutSep-Pakpurification,butsaltcontentprohibitsinjectionofmorethan50nmol.FurThermore,yieldstendtobelowerifthesampleisnotdesaltedpriortoHPLCinjection.
免责声明 本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容。
版权声明 未经蚂蚁淘授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘”。违反上述声明者，本网将追究其相关法律责任。
