【Luvena Fortuna/英国小木马G8537女童装】 Luvena Fortuna ...相关交流-蚂蚁淘在线

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/VisDetectorLC295

1.Equilibratecolumninbuffersystem,3mL/minflowrate(generalpurgemethod)

1.1.5minlowsaltbuffer(LSB)orA1.2.2mingradientto100%highsaltbuffer(HSB)orB1.3.5minHSBorB1.4.2mingradientto60%LSBorA1.5.5min60%LSBorA

2.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%LSB
FinalPreparativeProcedures
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.A_560/660/A₂₆₀ratiosshouldbearound17forcy3and25forcy5.

Expectedoverallyields:cy3:50-70%;cy5:45-65%.

FinalConsiderations

Currently150nmolofCy-dUTPcontainedina10mMmixtureoflabeledandunlabeleddNTPsisloadedontothecolumnlistedabove,dependingonthecontentsandconcentrationofthefiltrate;PCRflow-throughsamplestendtobecleanerandmayallowforlargerinjectionlots.Thus,theflow-throughfromPCRandRTreactionsmaybekeptseparatesothattheycanbeprocessedseparately.TheSep-Pakpurificationdesaltsthesampleandislikelytoremoveotherimpuritiespresentinthesample.HPLCpurificationcanstillbeperformedwithoutSep-Pakpurification,butsaltcontentprohibitsinjectionofmorethan50nmol.FurThermore,yieldstendtobelowerifthesampleisnotdesaltedpriortoHPLCinjection.

================ 蚂蚁淘在线 ================

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