Description:
OneTaq®RT-PCRKitcombinestwopowerfulmixes,M-MuLVEnzymeMixandOneTaqHotStart2XMasterMixwithStandardBufferfor2-stepRT-PCRapplications.ThetwomixesrequireminimalhandlingduringreactionsetupandyetofferconsistentandrobustRT-PCRreactions.
ThefirststrandCDNAsynthesisisachievedbyusingtwooptimizedmixes,M-MuLVEnzymeMixandM-MuLVReactionMix.M-MuLVEnzymeMixcombinesM-MuLVReverseTranscriptaseandmurineRNaseInhibitorwhileM-MuLVReactionMixcontainsdNTPsandanoptimizedbuffer.Thekitalsocontainstwooptimizedprimersforreversetranscriptionandnuclease-freewater.Ananchoredoligo-dTprimer[d(T)23VN]forcestheprimertoannealtothebeginningofthepolyAtail.TheoptimizedRandomPrimerMixprovidesrandomandconsistentprimingsitescoveringtheentireRNAtemplatesincludingbothmRNAsandnon-polyadenylatedRNAs.
TheamplificationstepfeaturesaOneTaqHotStartDNAPolymeraseinamastermixformat.OneTaqHotStartDNAPolymeraseoffershigherfidelitythanTaqandbetteramplification.RT-PCRproductupto6kbcanbegenerated(Figure1).
Figure1.FirstStrandDNASynthesis
FirststrandcDNAsysthesiswascarriedoutinthepresenceof1XM-MuLVEnzymeMixat42°Cusing0.5μgofhumanspleentotalRNAinthepresenceofdT23VN(lanes1,4and7)orRandomHexamerMix(lanes2,5and8).No-RTcontrolswerelanes3,6and9.OneTaqHotStart1XMasterMixwasusedtoamplifya1.5kbfragmentofbeta-actingene,a0.6kbfragmentofGAPDHgene,anda5.5kbfragmentfromp532genein35cycles.TheMarkerlane(M)contains2-LogDNALadder(NEB#N3200).Figure2.FirstStrandDNASynthesis
KitComponents
Thefollowingreagentsaresuppliedwiththisproduct:
| Storeat(°C) | Concentration |
Oligod(T)23VN | -20 | 50μM |
Nuclease-freeWater | -20 | |
M-MuLVEnzymeMix | -20 | 10X |
M-MuLVReactionMix | -20 | 2X |
RandomPrimerMix | -20 | 60μM |
OneTaq®HotStart2XMasterMixwithStandardBuffer | -20 | 2X |