Description:
TheWarmStartLAMPKit(DNA&RNA)isdesignedtoprovideasimple,one-stepsolutionforLoop-MediatedIsothermalAmplification(LAMP)ofDNAorRNA(RT-LAMP)targets.LAMPandRT-LAMParecommonlyusedisothermalamplificationtechniquesthatprovidesrapiddetectionofatargetnucleicacidusingLAMP-specificprimers(suppliedbytheuser)andastrand-displacingDNApolymerase.ThiskitissuppliedwiththeWarmStartLAMP2XMasterMix,whichcontainsablendofBst2.0WarmStartDNAPolymeraseandWarmStartRTxReverseTranscriptaseinanoptimizedLAMPbuffersolution.BothBst2.0WarmStartDNAPolymeraseandWarmStartRTxReverseTranscriptasehavebeenengineeredforimprovedperformanceinLAMPandRT-LAMPreactions.Afluorescentdyeisalsosuppliedtoenablereal-timefluorescencemeasurementofLAMP.TheWarmStartLAMPKitiscompatIBLewithmultipledetectionmethods,includingturbiditydetection,real-timefluorescencedetection(whenusedwithLAMPfluorescentdye)andend-pointvisualization.
NEB’sWarmStartLAMPKit(DNA&RNA)iscompatiblewithmultipledetectionmethods

TheNEBWarmStartLAMPKit(DNA&RNA)includesseparatefluorescentdyeforreal-timefluorescencemeasurement.Alternately,detectioncanbeaccomplishedbyturbiditydetectionorendpointvisualization.
NEB’sWarmStartLAMPKit(DNA&RNA)offersspeedandrobustsensitivity

ARNAtarget(HMBS2)wasamplifiedfromJurkattotalRNAusingtheWarmStartLAMPKitandOptigeneMasterMix(ISO-001).Reactionswereperformedat65°Cfor74minutesonareal-timeThermocycler(Bio-RadCFX96)intriplicate.Timetoresultissetasthetimeatwhichthefluorescencecrossedathresholdof10%ofmaximalfluorescence.NEB’sWarmStartLAMPKitresultedinfasterandmorerobustdetectionascomparedtotheOptiGeneMasterMix.
NEB’sWarmStartLAMPKit(DNA&RNA)providesfastLAMP/RT-LAMPresults

ExperimentsweredesignedtoamplifyaDNAtarget(BRCAb)andaRNAtarget(HMBS2)byLAMPorRT-LAMP,respectively.A25μlreactioncontaining10ngHeLaDNAor5ngJurkattotalRNA,1XMasterMix,1XLAMPprimersandfluorescentdyewasincubatedonaBio-RadCFX98instrumentat65°Cfor75minutes.Lucigen?OmniAmp?RNA&DNAKitandOptiGeneIsothermalMasterMix(ISO-001)wererunaccordingtomanufacturers’recommendations.NEBWarmStartLAMPKit(DNA&RNA)enablesspecificamplificationacrossmultiplespecies/targets
| PRIMERSET | DNATEMPLATE | 
|---|
| HeLa?DNA
 | Lyme?DNA
 | B.malayi?DNA
 | E.coliDNA
 | C.elegans?DNA
 | 
|---|
| BRCAb(human) | + | ? | ? | ? | ? | 
| CFTR(human) | + | ? | ? | ? | ? | 
| LymO(Lyme) | ? | + | ? | ? | ? | 
| BmHhal(B.malayi) | ? | ? | + | ? | ? | 
| dnaE(E.coli) | ? | ? | ? | + | ? | 
| Lec10b(C.elegans) | ? | ? | ? | ? | + | 
| Lec6(C.elegans) | ? | ? | ? | ? | + | 
Ineachindividualreaction,multiplegDNAtemplateswereaddedalongwithprimersspecifictoatargetinonlyonespecies.Reactionswereincubatedat65°Cfor74minutesonathermocycler(Bio-RadCFX96)intriplicate.Positiveresults(+)wereobservedonlyintheexpectedspecies/targetcombinations,demonstratinghighspecificityoftheWarmStartLAMPreactions.
KitComponents
Thefollowingreagentsaresuppliedwiththisproduct:
|  | Storeat(°C) | Concentration | 
| WarmStartLAMP2XMasterMix | -20 |  | 
| LAMPFluorescentDye(50X) | -20 |  |