诺唯赞试剂说明书 HiScript- 1st strand cDNA Synthesis Kit重翻

1、HiScript 1st strand cDNA Synthesis KitCatalog # R111IntroductionHiScript 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 50C. It can tolerate 55C reaction temperature and is ap

2、plicable 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 esp

3、ecially 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

4、 Transcriptase, 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. 2 RT Mix contains optimized buffer system and dNTP. This Mix contains HiScript Reverse Transcrip

5、tase and RNase inhibitor. Oligo (dT)18 and Random hexamers can be chosen as primers for reverse transcription as needed. Package InformationComponents R111-01 25 rxn (20l /rxn) R111-02 50 rxn (20l /rxn)RNase free ddH2O 1 ml1 ml2 RT Mix a250l500lHiScript Enzyme Mix b50l100lOligo (dT)18 (50M)25l50lRan

6、dom hexamers (50 ng/l)25l50la Contain 1 mM each dNTP. b Contain RNase inhibitor.StorageStore 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 dT18 as primer, rea

7、ct 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 at 50. Take

8、1/10 of cDNA products to carry out PCR amplification of -actin gene. After agarose gel electrophoresis and EB staining, a clear 550 bp band can be detected.ProtocolMaterials needed to be prepared by usersRNase free microcentrifuge tube of 1.5 ml or 0.2 ml PCR tubeWater bath or PCR instrumentIceRNAHi

9、gh-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 g total RNA.RNase inhibitor is already contained in HiScript Enzyme Mix, which is used to resist trace RNase that might

10、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 of 1st strand cDNA c

11、an be carried out by choosing Random hexamers, Oligo (dT) 18 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 structure or is GC-rich, and

12、Oligo (dT) 18 or gene specific primers (GSP) cannot effectively synthesize cDNA, Random hexamers can be used. Oligo (dT) 18 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 highest consistency can b

13、e obtained. Gene specific primer (GSP) has the highest specificity. But under some circumstances, GSP used for PCR reaction cannot effectively synthesize cDNA. Then use Oligo (dT) 18 instead and try again. Guidelines for PCRProducts of 1st strand cDNA can be directly used as templates for PCR reacti

14、on. It is suggested that the volume of cDNA as templates should not exceed 1/10 of the volume of PCR reaction. In accordance with different experimental objectives, the following DNA Polymerase is recommended:AceTaq DNA Polymerase. It is a chemically modified hot-start Taq enzyme with the highest se

15、nsitivity, and is recommended to amplify low-copy gene from cDNA. Phanta Super Fidelity DNA Polymerase. It is a high-fidelity DNA polymerase with the highest fidelity and extremely high amplification efficiency, and is recommended to amplify DNA for cloning. LAmp DNA Polymerase. It is recommended to

16、 perform long fragment PCR, and the amplicon length can reach up to 15kb. Guidelines for reverse transcription reactionSet up the following mixture in RNase free centrifuge tubeRNase free ddH2Oto 20 l2 RT Mix10 lHi Script Enzyme Mix2 lOligo (dT) 18 (50 M)1 or Random hexamers (50 ng/l)or Gene Specifi

17、c Primers (2 M)Template RNATotal RNA: 100 pg-5 g Poly (A)+ RNA: 10 pg-500 ngCarry out the first strand cDNA synthesis according to the following conditions:Use Oligo (dT)18 5045 min*855 minUse Random hexamers2510 min5045min*855minUse Gene Specific Primers50-5545min*855min*May be adjusted within 30-6

18、0 min. Prolonging reaction time may help to obtain longer cDNA (5 kb).HiScript Reverse Transcriptase can be deactivated by incubation at 85 for 5 min. cDNA can be stored at -20 or be immediately used in PCR reaction. TroubleshootingProblems and CausePossible solutionsNo PCRproductsRNase contaminatio

19、nBesides using RNase free tip and centrifuge tube, it is recommended that the reagents used are exclusive for RNA experiments and the operations should be performed at independent and clean area. Make sure to wear gauze masks and disposable gloves and avoid talking. After total RNA is extracted, it

20、is recommended to take a small amount to carry out electrophoresis on 1% agarose gel to confirm the integrity of RNA. Taking mammalian cell/tissue as example, if total RNA is intact, three bands can be seen clearly on gel, whose molecular weights are 28s, 18s and 5s respectively. If three bands can

21、be seen but their shapes are blurred or diffuse, then part of RNA degrades. In this case, please start reverse transcription immediately and properly increase the amount of templates; if only one band with small molecular weight or no band can be seen, RNA has been completely degraded. In this case, prepare new RNA. There are inhibitors of reverse transcription reaction in RNAInhibitors of reverse transcription reaction include phenol, salt, SDS, EDTA, etc. It is suggested to carefully wash off RNA precipitation (Flick the bottom of the tu