First Strand cDNA Synthesis Kit

MB037-5G: 5G First Strand cDNA Synthesis Kit with gDNA Elimination Function

The 5G first strand cDNA synthesis kit with gDNA elimination function provides a fast and efficient procedure for reverse transcription and genomic DNA elimination in RNA samples. To obtain accurate results in RT-PCR and RT-qPCR, 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 5G first strand cDNA synthesis kit with gDNA elimination function combines genomic DNA elimination of RNA samples and first strand cDNA synthesis in one easy workflow to ensure accuracy of RT-PCR and RT-qPCR results. Since there is no need to perform a separate DNase digestion and re-purify your RNA samples, your time and costs are saved.

The 5G first strand cDNA synthesis kit with gDNA elimination function is an upgraded version of the 4G first strand cDNA synthesis kit with gDNA elimination function. It includes a new generation of reverse transcriptase, 5G Reverse Transcriptase, and the most suitable for reverse transcription optimization. Buffer further improves the efficiency of one-strand synthesis. The 5 × gDNA Removing Buffer in this kit can quickly remove genomic DNA contamination at 42°C for 2 min, ensuring more reliable follow-up results, and simplifying qPCR primer design without the need to design primers across introns. The kit contains single-component reverse transcription primers Oligo (dT)20 VN and Random hexamers. Users can flexibly choose reverse transcription primers for subsequent experiments according to their needs. This kit can synthesize full-length cDNA (up to 20 kb) for downstream experiments such as cloning, and can also synthesize highly uniform cDNA for qPCR quantification.

Features of the 5G first strand cDNA synthesis kit with gDNA elimination function:

* Higher reverse transcription efficiency and higher cDNA yields from all regions of RNA transcripts: based on the more efficient 5G Reverse Transcriptase.
* Flexible choice of primers: Different types of reverse transcription primers can be used flexibly for different experimental designs.
* Quick and complete removal of genome contamination by the 5 × gDNA Removing Buffer: to ensure more reliable follow-up results, and simplify the design of qPCR primers, without the need to design primers across introns.

Components of the 5G first strand cDNA synthesis kit with gDNA elimination function:

gDNA Removing Buffer (5X): Buffer for effective elimination of genomic DNA contamination in RNA samples
RT Buffer (10X): Buffer optimized for reverse transcription; Contains MgCl2, dNTPs, and stabilizers
5G Enzyme Mix: Mixture of reverse transcriptase, and RNase inhibitor.
Oligo (dT)20 VN Primer.
Random Hexamers.
RNase-free H2O: Ultrapure quality.

Storage:

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.

MB037-4G: 4G First Strand cDNA synthesis Kit with gDNA Elimination Function

The 4G first strand cDNA synthesis kit with gDNA elimination function includes a new generation of reverse transcriptase, 4G Reverse Transcriptase, and the most suitable for reverse transcription optimization. The 4G Reverse Transcriptase is a new reverse transcriptase obtained through in vitro molecular evolution technology on the basis of M-MLV (RNase H-) Reverse Transcriptase. Compared with the previous generation of Reverse Transcriptase, the 4G Reverse Transcriptase has further greatly improved thermal stability and is very suitable for reverse transcription of RNA templates with complex secondary structures. With multiple point mutations which further enhances template affinity and progress, the 4G Reverse Transcriptase has higher tolerance to common reverse transcription inhibitors, which greatly improves the ability to synthesize full-length cDNA.

The 4G first strand cDNA synthesis kit with gDNA elimination function contains all the components needed to synthesize high-quality first-strand cDNA. The product is suitable for subsequent PCR, qPCR and other experiments. 2 × RT Mix contains optimized buffer system and dNTP; 4G Enzyme Mix contains 4G Reverse Transcriptase and RNase inhibitor. The Oligo (dT)23 VN included in the kit has a stronger anchoring ability for Poly A+ mRNA than Oligo (dT)18, making reverse transcription more efficient. Users can choose Oligo (dT)23 VN, Random hexamers or gene-specific primers as reverse transcription primers according to their needs. The subsequent flexible and optional operation steps can not only synthesize full-length cDNA (up to 20 kb) for cloning, but also efficiently synthesize cDNA with uniform reverse transcription efficiency for each position of qPCR.

Features of the 4G first strand cDNA synthesis kit with gDNA elimination function:

* Based on the highly efficient 4G Reverse Transcriptase, the super temperature tolerance ensures that the complex secondary structure of RNA can be opened under high temperature conditions to obtain longer cDNA.
* A wide range of template starting amount: from 1 pg - 5 μg total RNA.
* Long fragment amplification: up to 15 kb or more.
* Anchored Oligo (dT)23 VN primer is designed for binding site anchoring with high specificity, ensuring the efficiency and success rate of first-strand cDNA synthesis.
* Different primers are selected for different downstream applications. The synthesized one-strand cDNA is widely used in molecular cloning, hybridization, PCR amplification and qPCR reaction, and so on.

Components of the 4G first strand cDNA synthesis kit with gDNA elimination function:

gDNA Removing Buffer (5X): Buffer for effective elimination of genomic DNA contamination in RNA samples
RT Buffer (2X): Buffer optimized for reverse transcription; Contains MgCl2, dNTPs, and stabilizers
4G Enzyme Mix: Mixture of reverse transcriptase, and RNase inhibitor.
Oligo (dT)20 VN Primer.
Random Hexamers.
RNase-free H2O: Ultrapure quality.

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.
* Primers are 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 cDNAs added (from the undiluted RT reaction) should not exceed 1/10 of the final PCR volume.

Quality Control:

First strand cDNA synthesis kit components are free of contaminating DNase and RNase. The kits are functionally tested using real-time RT-PCR with SYBR Green qPCR Master Mix for amplification of GAPDH cDNA derived from human universal RNA.

PCR enzymes:
* Taq DNA polymerase.
* High yield Taq DNA polymerase.
* Hot-start high-fidelity DNA polymerase.
* Hot start Taq DNA polymerase with chemical or antibody inhibitor, used for SYBR green or Taqman probe-based real time PCR (qPCR or quantitative PCR).

2x PCR master mixes:
* 2x PCR PreMix with dye (green, red, blue).
* 2x rapid PCR Master Mix with a green dye.
* 2x long PCR Master Mix with a blue dye.
* 2x high yield PCR master mix with a blue dye.
* 2x hot-start high-fidelity PCR master mix with a blue dye.
* Blood direct PCR kit
* Cell and tissue direct PCR kit
* RT-PCR master mixes
* 2x qPCR master mixes
* RT-qPCR master mixes

PCR components and related products:
* dNTP mix, 10 mM each.
* dNTP mix, 25 mM each.
* Proteinase K powder .
* Proteinase K solution.
* Reverse Transcriptases.
* First strand cDNA synthesis kits.
* Murine RNase inhibitor.
* PCR enhancer.