MCLAB/RCA DNA Amplification Kit/PPK-100/1 Ea

Description:
RCADNAamplificationkitsarenovelproductsdevelopedspecificallytopreparetemplatesforDNAsequencing.AsillustratedinFigure1,theRCAmethodutilizesbacteriophagephi29DNApolymerasetoexponentiallyamplifysingle-ordouble-strandedcircularDNAtemplatesbyrollingcircleamplification(RCA)^(1,2).ThisisothermalamplificationmethodproducesmicrogramquantitiesofDNAfrompicogramamountsofstartingmaterialinafewhours.

AmplificationinvitroofverysmallamountsoftemplateDNAeliminatestheneedforovernightcellcultureandconventionalplasmidorM13DNApurification.TheproofreADIngactivityofphi29DNApolymeraseensureshighfidelityDNAreplication.⁽³⁾

Thestartingmaterialforamplificationcanbeasmallamountofbacterialcellscontainingaplasmid,anisolatedplasmid,intactM13phage,oranycircularDNAsample.BacterialcoloniescanbepickedfromagarplatesandaddeddirectlytotheRCAreaction.Alternatively,microliterquantitiesofasaturatedbacterialcultureoraglycerolstockcanserveasstartingmaterial.Dependingonthesourceofstartingmaterial,amplificationiscompletedin4–18hoursat30˚Cwithnoneedforthermalcycling.TheproductoftheRCAreactionishighmolecularweight,double-strandedconcatemersofthecirculartemplate.

NotethatwhenstartingwithM13clones,theRCAproductisdouble-strandedDNAandcanbesequencedwithforwardandreverseprimers.DNAamplifiedbytheRCAmethodcanbeuseddirectlyincyclesequencingreactionswithoutanypurification.

Figure:

Figure1,SchematicdiagramoftheRCAprocess.RandomhexamerprimersannealtothecirculartemplateDNAatmultiplesites.Phi29DNApolymeraseextendseachoftheseprimers.WhentheDNApolymerasereachesadownstreamextendedprimer,stranddisplacementsynthesisoccurs.Thedisplacedstrandisrenderedsingle-strandedandavailabletobeprimedbymorehexamerprimer.Theprocesscontinues,resultinginexponential,isothermalamplification.


Figure2.
 

Components:

RCADNAAmplificationKit(100reactions)
Cat#:PPK-100
Phi29DNAPolymerase,20µl,(10,000units/ml)
2xMCRCAmix,1ml
CellLysisBuffer,1ml

RCADNAAmplificationKit(500reactions)
Cat#:PPK-200
Phi29DNAPolymerase,100µl,(10,000units/ml)
2xMCRCAmix,5ml
CellLysisBuffer,5ml

References:
1.Dean,F.etal.,GenomeResearch11,1095–1099(2001).
2.Lizardi,P.etal.,Nat.Genet.19,225–232(1998).
3.Estaban,J.A.etal.,J.Biol.Chem.268,2719–2726(1993).