ImproveYourPCRPerformancewithPCRboost.Simply.
- EnhanceyourmostchallengingPCRsamples
- SimpletoUse
- Reducesnumberofcycles
- UseexistingTaqpolymeraseandPCRprotocol
Overview
EnhancePCRPerformanceforDifficultSamples
PCRBoostisauniquereagentthatenhancesend-pointandreversetranscription-PCRperformancebyimprovingsensitivityandspecificityduringamplificationofgenomicDNAorRNAtemplates.Touse,simplyreplacethewaterinyourPCRwithPCRBoost.That’sall.ItisdesignedtoenhancetheamplificationofDNAwithouttheneedfortimeconsumingoptimizations.PCRBoostsuccessfullyamplifiesthermallydegradedDNAresultinginavisIBLeampliconthatcanbeusedfordownstreamapplicationssuchascloningandsequencing.
ProductData
SuccessfulamplificationofthermallydegradedDNAusingPCRBoost
Figure1:GenomicDNA(gDNA)fromhumanbloodwaspurifiedusingtheQIAamp®DNABloodMiniKit.,and500ngwasexposedto70°Cfor20h.1µl(~50ng)ofthermallydegradedDNAorcontrolDNAstoredat4°CwasusedastemplateforPCRreactions.Thereactionsweresetupusing2.5UTaqDNApolymerase(NEB),3μl10xThermopolreactionbuffer(NEB),0.5µldNTPs(10µMeachnucleotide),0.5µleachofhumanβ-actinforwardandreverseprimers.ThevolumeinthethermallydegradedDNAPCRreactiontubeswasbroughtupeitherinwaterorinPCRBoost.FollowingPCRamplification,10µlofeachPCRreactionwasrunonanagarosegel.A)500nghumangenomicDNAstoredat4°Candthermallydegradedat70°Cfor20hrunonanagarosegel.L:1kbladder.B)50ngofcold-stored(4°C)andthermallydegradedDNAusedastemplateinPCRreactionsamplifyingthehumanβ-actingene.PCRreactionswerebroughtuptovolumeeitherinwaterorPCRBoost.L:ladder.
SerialDilutionUsingPCRBoost
Figure-2:50nghumangenomicDNA(gDNA)wasamplifiedbyPCRusing2.5UTaqDNApolymerase(NEB),3µl10xthermopolreactionbuffer(NEB),0.5µldNTPs(10µMeachnucleotide),0.5µleachhumanβ-actinforward(5’ctacctcatgaagATCCtcacc3’)andβ-actinreverse(5’gtacttgcgctcaggaggagc3’;10µMeach)inafinalvolumeof30µl.ThePCRBoostvolumewasseriallydilutedandreplacedbywater*.Cyclingparameterswere:94°Cfor5minfollowedby40cyclesof94°Cfor15sec,55°Cfor30secand72°Cfor30sec.10µlofeachPCRreactionswasrunona0.8%agarose/ethidiumbromidegel.
IdenticalReactionsinWaterVersusPCRBoost
Figure-3:100,50,20,10or4nggDNAwasusedinPCRreactionswherethereactionwasbroughtuptovolumeineitherwaterorPCRBoost.Sampleswereusedtoamplifythefibroblastgrowthfactor13(FGF13)genebyPCRusing2.5UTaqDNApolymerase(NEB),3µl10xthermopolreactionbuffer(NEB),0.5µldNTPs(10µMeachnucleotide),FGF13forward(5’gAATgttaacaacatgctggc3’)andFGF13reverse(5’agaagctttaccaatgttttcca3’)(kindgiftofDr.D.Cohn)inafinalvolumeof30µl*.Cyclingparameterswere:94°Cfor5minfollowedby40cyclesof94°Cfor15sec,55°Cfor30secand72°Cfor30sec.10µlofeachPCRreactionwasrunona0.8%agarose/ethidiumbromidegel.
ComparisonofPCRBoosttoOtherPCREnhancers
Figure-4:PCRenhancersfrom3differentcompaniesweretestedalongsidePCRBoostandcomparedtoanon-enhancedwatercontrol*using1nggDNA(conditionsusedwerethesameasFigure2above).
UseofPCRBoostwithThreeDifferentTaqPolymerases
Figure-5:TaqDNApolymerasesfrom3differentsuppliersweretestedalongsidePCRBoostusing10nggDNA*(conditionsusedwerethesameasFigure2above).
TestingofPCRBoostinRT-PCR
Figure-6:RT-PCRreactionsweresetupusingthegenespecificreverseprimerforthesingle-copyRNasePgene(5’agaccatcctggctaacacg3’).SmallamountsoftotalRNA(100ng)extractedfrom293cells(humanadenocarcinomacellline)wereusedforfirststrandCDNAgenerationusingmanufacturer’sinstructionsfortheAffinityScript™(Stratagene/AgilentTechnologies)RT-PCRkit.ReactionsweresetupusingeitherwaterorreplacingthewatercomponentwithPCRBoost.1µlofthecDNAwasthenusedinasubsequentsecondstrandPCRreactionusingforward(5’ttcactgcttcatgcctacg3’)andreverseprimersfortheRNasePgene(conditionsusedwerethesameinFigure2above).ReactionsweresetupusingeitherwaterorreplacingthewatercomponentwithPCRBoost.
*PCRreactionsweresetupusingcocktailsofcommonelements.
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Specifications
- Productformat: 1mL
- Storagetime: 6months
- Storagetemperature: 15–25°C
- Downstreamapplications:plasmidpurification,bacterialpropagation,glycerolstockpreparation
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