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Useofoligonucleotidesinvariousresearchapplicationsrequirescertainbasicstorageandhandlingtechniquesinordertoensuretrouble-freeexperiments.Properstorageofyouroligonucleotidewillmaximizeitsshelf-life,allowingyoutogetthemostusefromyouroligo.Followingthesesimpledilutionandweight/volumeunitconversionguidelineswillenableyoutoworkeasilywithyourSigma-Genosysoligos. WhatshouldIusetoresUSPendmyoligo? Oligonucleotidesmayberesuspendedineithersterilewateror,preferably,TE(10mMTris,pH7.5-8.0,1mMEDTA).TheuseofDEPC-treatedwaterisnotrecommended.IfyouaremakingaconcentratedstocksolutionwerecommenddissolvingyouroligoinTE.Workingsolutionscanbedilutedfromthestocksolutionusingsterile,nuclease-freewater. HowshouldIstoremyoligo? AllSigma-Genosysoligosareprovideddesalted,dry,andreadyforuseuponresuspension.Onceresuspended,oligonucleotidestocksolutionsarebestkeptfrozenat-20篊forseveralweeksandmayremainstableforseveralmonths.Themostimportantfactorinstoringworkingsolutionsisusingnuclease-free,sterilewater.Forlong-termstorageofoligonucleotidestocks,werecommendthatyoudrydownthesamplesandstoreat-20篊.OligonucleotidesmodifiedwithfluorescentdyesshouldbekeptawayfromlightasmuchaspossIBLe.Alkalinephosphatasemodifiedoligonucleotidesshouldbestoredat4篊,neverfrozen. HowdidSigma-Genosysquantifymyoligo? ThemicrogramquantityofoligonucleotidewesupplyisdirectlyrelatedtoitsUVopticaldensity(O.D.)atawavelengthof260nm.Sigma-GenosystakesgreatcaretoensurethattheO.D.reADIngdeterminedforeacholigonucleotideisasaccurateaspossible.Afterdesaltingandfurtherpurificationofyouroligoweresuspenditin200ulofwater.Weremovea6ulaliquot(toensurewearetakingavolumewithintheaccurateandreproduciblerangeofmicroPipettes)fromthesuspensionanddiluteitwith994ulofsterilewaterrequiredtodeterminetheO.D.usingaspectrophotometer.WeensurethattheO.D.readingiswithinthelinearrangeofaccuracyofourspectrophotometers.Anysamplenotwithin0.1and1.5O.D.unitsisremeasuredusingamoreappropriatedilutionforthatsample. Becausewetakeonly6ulfromtheoriginal200ulresuspension,theO.D.ofthe6ulmustbemultipliedbyafactorof33(whichis200/6)todeterminetheO.D.ofthewholeresuspension.Ingeneral,itcanbeassumedthat1O.D.unitisequivalentto30ug,whichisareasonableaverageformostoligonucleotides.Therefore,thetotalnumberofmicrogramsofoligointhe200ulresuspensioncanbecalculatedasfollows: Totalugofoligoinoriginal200ul = O.D.readingforX33X30ug6ulaliquot Sigma-Genosysaccuratelydeterminestheextinctioncoefficientforeacholigo(seeQuantitatingDNA)Thequantityofoligonucleotidesenttoyou(seeYields)isexpressedinnanomolesonyourtubelabel,alongwiththeugquantityoftheoligoanditsmolecularweight(MW).Wecalculatethenumberofnanomolesusingtheamountofoligo(inug)andthemolecularweightasfollows: Totalnmoleoligo = ugofoligo X 1umol X 1000nmole MW 1micromole = ugofoligo X 1000 MW (Note:1mole=10e3mmoles=10e6micromoles=10e9nanomoles=10e12picomoles) SampleResearchApplication "Mytubecontains35.22nmolesofoligo,butIneedafinaloligonucleotideconcentrationof0.5uMinmyreaction.Myreactionvolumeis100ul.HowmucholigodoIaddtomyreaction?" First,makeaconcentratedoligonucleotidestocksolution(10Xor100X,dependingontheconcentrationyouneed).Thenaddanappropriatevolumetothereaction,therebyachievingthedesiredworkingconcentration.Serialdilutionsofthestock(s)mayberequiredtoensurepipettingaccuracy.Seeoutlinedexamplebelow: 1.Decideonastockconcentrationandstandardizeyourunits. Desiredfinalconcentration=0.5uM 100Xstockconcentration=100X0.5uM=50micromole/liter Totalnanomolesofoligo=35.22nmole(seetubelabelforyouractualoligo) Totalmicromolesofoligo=35.22/1000=0.03522micromole Note:Thequantityofoligomustbeexpressedinmicromolestocalculatethevolumeforamicromolarsolution. 2.Makea100Xstocksolution(100X0.5uM=50uM). Whatvolume(V)isrequiredtoresuspend0.03522micromoleofoligotogiveastockconcentrationof50uM?Usethefollowingstepsasaguide: 50micromole = 0.03522micromole SolveforV 1000ml Vml V = 0.03522x1000m 50 V=0.704ml=704ul 3.Useserialdilutionstomakea10Xstock(10X0.5uM=5uM). Forpipettingaccuracy,avoidusingsmallvolumes(lessthan2ul)ofstocksolutionsinyourreactions.Itmaybenecessary,therefore,toseriallydiluteyourstockoligonucleotidesolution.Makea10Xstockbydilutingyour100Xstockasfollows: 1:10dilutionof100Xstock 10ulof50uM(100X)stock+90ulsterilewater(orTE)=100ulof5uM(10X)stock 4.Usea10Xstocktogivethefinalconcentrationinthereaction. Myreactionvolumeis100ul.Howmuchofthe10X(5uM)stocksolutionofoligoisrequiredtoyielda0.5uMconcentrationofoligointhetotalreaction? Vol.of(10X)stockXstockconc.=Finalreactionvol.Xfinaloligoconc. (Sul)X(5uM)=(100ul)X(0.5uM)SolveforS S = 100X0.5ul 5 S=10ul PDF文档下载： 
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