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HiScript® 1st strand cDNA Synthesis Kit Catalog # R111 Introduction HiScript® 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°C. It can tolerate 55°C reaction temperature and is applicable 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 especially 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 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 Transcriptase and RNase inhibitor. Oligo (dT)18 and Random hexamers can be chosen as primers for reverse transcription as needed. Package Information Components R111-01 25 rxn (20μl /rxn) R111-02 50 rxn (20μl /rxn) RNase free ddH2O 1 ml 1 ml 2 × RT Mix a HiScript™ Enzyme Mix b Oligo (dT)18 (50μM) Random hexamers (50 ng/μl) a Contain 1 mM each dNTP. b Contain RNase inhibitor. 250μl 50μl 25μl 25μl 500μl 100μl 50μl 50μl Storage Store at -20℃. Quality Control None 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, 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 at 50℃. Take 1/10 of cDNA products to carry out PCR amplification of β-actin gene. After agarose gel electrophoresis and EB staining, a clear 550 bp band can be detected. Protocol Materials needed to be prepared by users RNase free microcentrifuge tube of 1.5 ml or 0.2 ml PCR tube Water bath or PCR instrument Ice RNA 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 μg total RNA. RNase inhibitor is already contained in HiScript™ Enzyme Mix, which is used to resist trace RNase that might exist in the environment or system. Synthetic condition of 1st strand cDNA All 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 primers Synthesis of 1st strand cDNA can 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 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 be 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 PCR Products of 1st strand cDNA can be directly used as templates for PCR reaction. 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 sensitivity, 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 perform long fragment PCR, and the amplicon length can reach up to 15kb. Guidelines for reverse transcription reaction Set up the following mixture in RNase free centrifuge tube RNase free ddH2O to 20 μl 2 × RT Mix 10 μl Hi Script™ Enzyme Mix 2 μl Oligo (dT) 18 (50 μM) 1μ or Random hexamers (50 ng/μl) or Gene Specific Primers (2 μM) Total RNA: 100 pg-5 μg Poly (A)+ RNA: 10 pg-500 ng Template RNA Carry out the first strand cDNA synthesis according to the following conditions: Use Oligo (dT)18 50℃ 45 min* 85℃ 5 min Use Random hexamers 25℃ 10 min 50℃ 45min* 85℃ 5min Use Gene Specific Primers 50-55℃ 45min* 85℃ 5min *May be adjusted within 30-60 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. Troubleshooting Problems and Cause Possible solutions No PCRproducts RNase contamination There are inhibitors of transcription reaction in RNA Besides 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 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 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. reverse Inhibitors of reverse transcription reaction include phenol, salt, SDS, EDTA, etc. It is suggested to carefully wash off RNA precipitation (Flick the bottom of the tube to suspend the precipitation, and leave it still for several minutes) with 75% ethanol (set up with DEPC water). There are complex secondary structures or high GC content in the target area of RNA. Use Random hexamers as primers for 1st strand synthesis. Elevate the reverse transcription temperature to 55℃. Non-specific amplification Contamination of genomic DNA Use Trizol total RNA extraction reagent (such as RNA isolater, cat No. R401-01), add chloroform and separate it by centrifuging. Avoid pipe the middle layer when transfer the supernatant. Use primers that hybridize both contiguous exons. PCR conditions are not optimized Use hot-start Ace TaqTM DNA Polymerase Adjust annealing temperature and concentration of Mg2+