诺唯赞试剂说明书 HiScript- 1st strand cDNA Synthesis Kit重翻.docx
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1、HiScript 1st strand cDNA Synthesis KitCatalog # RillIntroductionHiScript Reverse Transcriptase is a brand new reverse transcriptase based on mutagenesis of M-MLV (RNase H-) Reverse Transcriptase. HiScript Reverse Transcriptase is most active at 50. It can tolerate 55 reaction temperature and is appl
2、icable to reverse transcription of RNA templates with secondary structures. Elevated affinity of HiScript Reverse Transcriptase to the template makes reverse transcription reaction more efficient, with more full-length cDNA can be obtained and as low as 1 pg total RNA can be detected, which is espec
3、ially applicable to a small amount of templates and the reverse transcription of low-copy genes. In addition, the mismatch rate of HiScript Reverse Transcriptase is lower than that of M-MLV (RNase H-) Reverse Transcriptase, thereby increasing the fidelity of cDNA cloning.Based on HiScript Reverse Tr
4、anscriptase, HiScript 1st strand cDNA Synthesis Kit contains all components needed to synthesize high-quality 1st strand cDNA. Reaction products are applicable to subsequent PCR, qPCR and PCR cloning.2x RT Mix contains optimized buffer system and dNTP. This Mix contains HiScript Reverse Transcriptas
5、e and RNase inhibitor. Oligo (dT)is and Random hexamers can be chosen as primers for reverse transcription as needed.Package Informationa Contain 1 mM each dNTP. b Contain RNase inhibitor.ComponentsRlll-01 25 rxn (20l1 /rxn)Rlll-02 50 rxn (20gl /rxn)RNase free ddHzO1 ml1 ml2xRT Mix a250|il500pilHiSc
6、ript Enzyme Mix b50|il100 fliOligo (dT)i8 (50|iM)25 m50|ilRandom hexamers (50 ng/|il)25 ul50MStorageStore at -20.Quality ControlNone of the components is contaminated by exonuclease, exonuclease or RNase after testing.Functional test 1: Taking 500 ng Hela cell total RNA as template and Oligo dT 18 a
7、s primer, react for 30 min at 50. Take 1/10 of cDNA products to carry out PCR amplification of DNCH gene. After agarose gel electrophoresis and EB staining, a clear 5.6 kb band can be detected.Functional test 2: Taking 100 pg Hela cell total RNA as template and Oligo dT18 as primer, react for 30 min
8、 at 50. Take 1/10 of cDNA products to carry out PCR amplification of p-actin gene. After agarose gel electrophoresis and EB staining, a clear 550 bp band can be detected.ProtocolMaterials needed to be prepared by users RNase free microcentrifuge tube of 1.5 ml or 0.2 ml PCR tubeWater bath or PCR ins
9、trument IceRNA High-quality and integrate RNA is of critical importance to obtaining high-quality cDNA. HiScript 1st strand cDNA Synthesis Kit is applicable to reverse transcription of 1 pg to 2.5 (ig total RNA. RNase inhibitor is already contained in HiScript Enzyme Mix, which is used to resist tra
10、ce RNase that might exist in the environment or system.Synthetic condition of 1st strand cDNAAll operations should be performed on ice. For GC-rich templates or those with complex secondary structures, the temperature for synthesis of cDNA 1st strand can be increased to 55 .Choosing primersSynthesis
11、 of 1st strand cDNA can be carried out by choosing Random hexamers, Oligo (dT) is or gene specific primer (GSP) as primers. Random hexamers is of lowest specificity. All RNA, including mRNA, rRNA and tRNA can be templates of Random hexamers. When the target area of RNA has complex secondary structur
12、e or is GC-rich, and Oligo (dT) 18 or gene specific primers (GSP) cannot effectively synthesize cDNA, Random hexamers can be used. Oligo (dT) is hybridizes at high efficiency to the 3 poly (A) region present in most mature eukaryotic mRNA. It is the first choice for most cases and generally the high
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