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1 µg of total mouse heart RNA and oligo(dT)18 were used in the reverse transcription reaction with RevertAid H Minus First Strand cDNA Synthesis Kit.
Synthesized cDNA was used as a template in subsequent PCR with Long PCR Enzyme Mix. A PCR primer specific to the 5’-end of mouse dystrophin gene mRNA and a series of flanking primers were used to amplify different regions of the gene.
M1 – GeneRuler DNA Ladder Mix1-6 – RT-PCR productsM2 – Lambda – pUC Mix Marker
Thermo Scientific RevertAid H Minus First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from RNA templates. The kit includes RevertAid H Minus Reverse Transcriptase, which has a point mutation that completely eliminates RNase H activity. Therefore, it does not degrade RNA in RNA-DNA hybrids during synthesis of the first strand cDNA.
The RevertAid H Minus First Strand cDNA Synthesis Kit contains RevertAid H Minus Reverse Transcriptase, RiboLock RNase Inhibitor, 5X Reaction Buffer, dNTP Mix, Oligo(dT)18 Primer, Random Hexamer Primer, Control RNA, Control Primers and nuclease-free water.
The recombinant RiboLock RNase Inhibitor, supplied with the kit, effectively protects RNA template from degradation. It is fully compatible with the reverse transcription reaction, as it maintains activity at temperatures up to 55°C.
The kit is supplied with both oligo(dT)18 and random hexamer primers. The oligo(dT)18 primer anneals selectively to the poly(A) tail of mRNA. Random hexamer primers do not require the presence of poly(A), therefore, they can be used for transcription of the 5’-end regions of mRNA or for cDNA synthesis using RNA without the poly(A) tail, e.g., microRNAs. Gene-specific primers may also be used with the kit. The first strand cDNA can be directly used as a template in PCR, realtime PCR or in second strand cDNA synthesis.
A. Schmidt, Y. H. Su et al., UPS1 and UPS2 from Arabidopsis Mediate High Affinity Transport of Uracil and 5-Fluorouracil. J. Biol. Chem. 279(43), 44817-44824 (2004).
K. G. Papavinasasundaram et al., Deletion of the Mycobacterium tuberculosis pknH Gene Confers a Higher Bacillary Load during the Chronic Phase of Infection in BALB/c Mice. J. Bacteriol. 187(16), 5751-5760 (2005).