ARNaseH-reversetrancriptasewithimprovedthermalstABIlity,efficiencyandprocessivityfortheproductionofCDNAfromlonganddifficultmRNAtemplates.
- Improvedthermalstability:Activeupto85ºC
- HighyieldsoffirststrandcDNA
- Highprocessivity:ProducescDNAoflongestmRNAsinthemammaliantranscriptome
- Greatsensitivity: WorkswithpgamountsofstartingtotalRNA
- Highreproducibility
- Robustandreliable
SunScript™ReverseTranscriptaseRNaseH-isaengineeredreversetranscriptasebasedonthewellcharacterizedHIV-1RTwithhighThermostabilityandnoRnaseHactivity.IthasRNA-andDNA-dependentpolymeraseactivity.Thisenzymeisactiveupto85°C.TheeliminationofRNaseHactivityenablesthisenzymetoproducehighyieldsoffulllengthcDNAfromlongRNAmolecules,andthehighthermalstabilitypromotesreversetransriptionthroughcomplexsecondarystructures.
- Reversetransciptionattemperaturesashighas85°C
- HighsensitivityoverawideRNAofstartingRNAamounts
- ReversetranscriptionoflongmRNAs
- HighcDNAsynthesisrate
Reversetransciptionattemperaturesashighas85°C
100ngoftotalRNAfromHeLacellswerereversetranscribedattheindicatedtemperaturesfor60minwithSunScriptReverseTranscriptasesandOligo(dT)18 primer,followedbyinactivationat95°Cfor10min.PresenceofGAPDHcDNAwasdetectedbyPCR(productlength305bp).
HighreactiontemperaturesareadvantageoustotranscribeRNAswithcomplicatedsecondarystructures.
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HighsensitivityoverawiderangeofRNAconcentrations
Sensitivitytest:RTreactionswereperformedat70°Cfor60min.Inactivationat95°Cfor10min.
Primerset:HumanGAPDH305bpset
1-1µgHumantotalRNAfromHeLacells
2-100ngHumantotalRNAfromHeLacells
3-10ngHumantotalRNAfromHeLacells
4-1ngHumantotalRNAfromHeLacells
5-100pgHumantotalRNAfromHeLacells
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TranscriptionoflongmRNAs
1-SunScript™RTRNaseH+replicate1
2-SunScript™RTRNaseH+replicate2
3-SunScript™RTRNaseH+replicate3
4-SunScript™RTRNaseH-replicate1
5-SunScript™RTRNaseH-replicate2
6-SunScript™RTRNaseH-replicate3
AssayforlongmRNAreversetranscription:3independentRTreactionswereperformedat65°Cfor60minusingOligo(dT)20 primers.Inactivationat95°Cfor10min.PCRPrimersets:mmNEB(>16kbfrom3’endofmRNA;mRNAtotallength22,5kb);mmDnah8(>8,5kbfrom3’endofmRNA;mRNAtotallength14,5kb)
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HighcDNAsynthesisrate
1µgoftotalRNAfromHeLacellswasreversetranscribedat65°CfortheindicatedtimeswithSunScript™ReverseTranscriptasesandOligo(dT)18primer,followedbyinactivationat95°C10min.
PCRPrimerset:hsHERC4033(>11kbfrom3’endofmRNA;mRNAtotallength15.3kb)
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ORDERINFORMATION
EachSunScript™RTRNaseH-contains:enzyme,5XReactionBufferandDTT.ThecompletemanualisavailableundertheManualtab.LucigenisanauthorizeddistributorofSygnisproductsintheUS.
SunScript™ReverseTranscriptaseRNaseH-isintendedformolecularBIOLOGyuseonlyandinvitrouseonly.Thisproductisnotintendedfordiagnosis,preventionortreatmentofadiseaseinhumanbeingsoranimals.
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我在做5RACE,选用TaKaRa公司的RNA反转录酶(RNaseH-),不知道这个酶好不好,有什么需要注意的。
2、取灭过菌且无核酸酶的0.2ml离心管,依次加入2~5μgRNAnμL
3、65℃保温5min,然后冰浴5min;
4、往3步骤中的0.2ml离心管依次加入下列组份
RNase抑制剂(40u/μL)0.5μL
10×M-MLVReactionBuffer2μL
DTT(200mM)1μL
逆转录酶(M-MLV)1μL
5、轻轻混匀后,然后2000rpm离心20s;
6、先在37℃保温1hr,然后70℃保温15min;
7、上述产物可立即进行下一步的PCR反应或-20℃保存。向左转|向右转
多谢……
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