Description:
Single-StrandBindingProtein(SSB)bindstosingle-strandedDNAwithhighaffinityandalsobindstoRNAanddouble-strandedDNAwithloweraffinity(1).Invivo,itstABIlizestransientlyformedssDNAandplaysanimportantroleinDNAreplication,recombinationandrepair(2).Invitro,SSBproteinshavebeenusedtodestabilizesecondarystructuresinDNAandtoincreasetheprocessivityofDNApolymerasesinseveralmolecularBIOLOGyapplications:SSBsimprovetheyieldandefficiencyofreversetranscriptionreactionsduringRT-PCRaswellasincreasetheyieldofPCRproducts(3-9).
ETSSB(ExtremeThermostableSSB)isasingle-strandedDNAbindingproteinisolatedfromahyperthermophilicmicroorganism,anditisaflagshipenhancerofthePrimerNavigatorproductseries.Itremainsfullyactiveafterincubationat95ºCfor60min.Duetotheextremethermostability,ETSSBcanbeusedinapplicationsthatrequireextremelyhightemperatureconditions,suchasnucleicacidamplificationandsequencing.
Source:
Applications:
ImprovetheyieldofmultiplexPCR(Fig.1)andmultiplexHDA(Fig.2)ImprovetheprocessivityofDNApolymerase(10)StabilizationandmarkingofssDNAstructure(11)IncreasetheyieldandspecificityofPCRreactions(5-9)IncreasetheyieldandprocessivityofRTduringRT-PCR(3,4)ImproveDNAsequencingthroughregionswithstrongsecondarystructure(8)EnhancetheRecAactivityforssDNAbindingandstrandtrasfer(12,13)
StorageConditions:
Concentration:
0.5mg/ml
StorageBuffer:
20mMTris-HCl
200mMNaCl
1mMEDTA
0.5mMDTT
50%Glycerol
pH7.5@RT
StorageTemperature:
-20ºC
QualityControl:
QualityAssuranceStatement:
ETSSBispurifiedfreeofcontaminatingendonucleasesandexonucleases.Eachlotistestedforsingle-strand,DNA-dependentATPaseactivityandisvisuallydeterminedtobe>95%pureonanSDS-polyacrylamidegel.
ExonucleaseActivity:
Incubationof20µgETSSBfor4hoursat37°Cin50µlreactionbuffercontaining50mMpotassiumacetate,20mMTris-acetate,10mMmagnesiumacetateand1mMdithiothreitol,pH7.9@25°C,with1µgofamixtureofsingleanddouble-stranded[3H]E.coliDNA(200,000cpm/µg)released<0.05%ofthetotalrADIoactivity.
EndonucleaseAssay:
Incubationof10µgETSSBfor4hoursat37°Cin50µlreactionbuffercontaining50mMpotassiumacetate,20mMTris-acetate,10mMmagnesiumacetateand1mMdithiothreitol,pH7.9@25°C,with1µgΦX174RFIDNAgave<5%conversiontoRFII.
NucleaseActivity:
Incubationof20µgETSSBfor16hoursat37°Cin50µlofreactionbuffercontaining50mMpotassiumacetate,20mMTris-acetate,10mMmagnesiumacetateand1mMdithiothreitol,pH7.9@25°C,with1µgλDNAyieldedaclearandsharpbandonanagarosegel.
References:
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