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First Strand cDNA Synthesis Kit
Optimal first strand cdna synthesis kit performance should be determined experimentally by the investigator.
MB037-11025: First strand cDNA Kit with gDNA elimination function
First Strand cDNA Kit, designed for use in two-step RT-qPCR, provides a fast and efficient procedure for reverse transcription and genomic DNA elimination in your RNA samples. To obtain accurate results in real-time RT-PCR, it is important that only cDNA is amplified and detected. Interference by genomic DNA can be avoided by designing primers or probes that span an exon/exon boundary. However, in cases where this is not possible (e.g., the cDNA is from a single-exon gene, or there exist processed pseudogenes in genomic DNA sequences), it is essential that the starting RNA sample is free of genomic DNA. The First Strand cDNA Kit combines genomic DNA elimination of RNA samples and subsequent first strand cDNA synthesis in one easy workflow to ensure accuracy of real-time RT-PCR results. Since there is no need to perform a separate DNase digestion and re-purify your RNA samples, your time and costs are saved.
* cDNA synthesis and genomic DNA elimination in only 20 minutes.
* No need to design RNA-specific primers.
* High cDNA yields from all regions of RNA transcripts.
gDNA Removing Buffer (4X): Buffer for effective elimination of genomic DNA contamination in RNA samples
RT Buffer (5X): Buffer optimized for reverse transcription; Contains MgCl2, dNTPs, and stabilizers
Enzyme Mix: Mixture of reverse transcriptase, and RNase inhibitor.
Primer Mix: Optimized mixture of random primers, and oligo-dT.
RNase-free H2O: Ultrapure quality
The kit is stable for one year when stored in a constant temperature freezer at -20°C. After thawing, mix the components thoroughly before using. Frequent freezing and thawing is not recommended.
Guidelines for Reverse Transcription:
* High-quality RNA is essential for high-quality cDNA synthesis.
* Set up all reactions on ice to minimize the risk of RNA degradation.
* After reverse transcription, the reaction must be inactivated by incubation at 95°C for 3 minutes.
* Primer Mix is supplied in this kit. If gene-specific primers (not supplied) should be used, we recommend using a final concentration of 0.7 uM, and a primer titration from 0.5 uM to 1 uM can be performed.
* For two-step RT-qPCR, the volume of the cDNA added (from the undiluted RT reaction) should not exceed 1/10 of the final PCR volume.
First Strand cDNA Kit components are free of contaminating DNase and RNase. The kit is functionally tested using real-time two-step RT-PCR with SYBR Green qPCR Master Mix for amplification of GAPDH cDNA derived from Human universal RNA.