Reverse Transcription

Product description This product, based on the classic reverse transcription reagents, has optimized the reaction time. The total reaction time can be as short as less than 6 minutes, while maintaining stable detection rates, specificity, and yield. It is suitable for reverse transcription of RNA templates that are complex, present in low amounts, or represent low-copy genes. The reverse transcription product of this product can be used for downstream PCR or qPCR applications. The kit provides two types of cDNA synthesis primers: Random Primers N6 and Oligo (dT)18. Users can choose according to their needs. Specifications Catalog Number N132064E N132064S Specifications 10 T 100 T Components Catalog Number Component Name N132064E N132064S N132064-A 4×Fast cDNA Synthesis Mix (No Dye) 50 μL 500 μL N132064-B 5×gDNA Digester Mix 20 μL 200 μL N132064-C Random Primers（50 μM） 20 μL 200 μL N132064-D Oligo d(T)18 Primers (50 μM) 20 μL 200 μL N132064-E RNase-free H2O 200 μL 2×1 mL Storage Store at -25 to -15℃. Valid for 1 year. Notes 1. All operations should be carried out on ice, and RNase contamination should be avoided during the process. 2. For your safety and health, please wear a lab coat and disposable gloves when operating. 3. For Research Use Only. Instructions 1. Reverse Transcription with gDNA Removal Step 1) gDNA Digestion Prepare the following mixture in an RNase-free centrifuge tube and gently mix by pipetting. Incubate at 42°C for 2 minutes. Component Usage Amount 5×gDNA Digester Mix 2 μL Total RNA or mRNA Total RNA：10 pg-5 μg mRNA：10 pg-500 ng RNase-free H2O Up to 10 μL 【Note】It is recommended that the input amount of Total RNA does not exceed 2 µg. If the expression level of the target gene is low, the input amount can be increased up to a maximum of 5 µg. 2) Reverse Transcription Reaction Setup Component Usage Amount The reaction mixture from the previous step 10 μL 4×Fast cDNA Synthesis Mix (No Dye) 5 μL Oligo d(T)18 Primers（50 μM) or Random Primers（50 μM） 2 μL RNase-free H2O Up to 20 μL 【Notes】 a. The amount of primer can be adjusted according to the amount of template input. If the downstream experiment is qPCR, Random Primers can be added to the reaction mixture at the recommended amount. b. It is recommended to first add the 4×Fast cDNA Synthesis Mix (No Dye) and mix thoroughly before adding the reverse transcription primer to ensure that the primer is not affected by the gDNA Digester. 3) Reverse Transcription Procedure Settings Temperature Time 55℃ 5 min 85℃ 5 sec 2. Reverse Transcription without gDNA Removal Step 1) Reverse Transcription Reaction Setup Component Usage Amount 4×Fast cDNA Synthesis Mix (No Dye) 5 μL Oligo d(T)18 Primers（50 μM) or Random Primers（50 μM） 2 μL Total RNA or mRNA Total RNA：10 pg-5 μg mRNA：10 pg-500 ng RNase-free H2O Up to 20 μL 2) Reverse Transcription Procedure Settings Temperature Time 55℃ 5 min 85℃ 5 sec 【Notes】 a. It is recommended that the input amount of Total RNA does not exceed 2 µg. If the expression level of the target gene is low, the input amount can be increased up to a maximum of 5 µg. b. The amount of primer can be adjusted according to the amount of template input. If the downstream experiment is qPCR, Random Primers can be added to the reaction mixture at the recommended amount. c. For templates with high GC content or complex structures, the reverse transcription temperature can be increased to 60°C. d. This product can synthesize cDNA sequences up to 14 kb in length. If longer cDNA products are required, the reverse transcription time can be appropriately extended.

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