Hifair™ III 1st Strand cDNA Synthesis Kit (gDNA digester plus) is a first-strand cDNA synthesis kit incorporating a genomic DNA (gDNA) removal step. This kit is based on Hifair™ III Reverse Transcriptase, which enables rapid cDNA synthesis and exhibits significantly enhanced thermal stability, tolerating reaction temperatures up to 60°C, making it ideal for reverse transcription of RNA templates with complex secondary structures. The enzyme also demonstrates improved template binding affinity, suitable for low-input RNA and low-abundance gene targets. Additionally, the Hifair™ III Reverse Transcriptase has enhanced capability to synthesize full-length cDNA, capable of producing cDNA up to 19.8 kb in length.

The kit includes a gDNA Digester Mix to remove residual genomic DNA contamination from RNA samples, ensuring more reliable downstream results. Two cDNA synthesis primers are provided: Random Primers N6 and Oligo (dT)18. Users may choose Random Primers N6, Oligo (dT)18, or gene-specific primers according to experimental needs. The resulting single-stranded cDNA can be directly used in downstream PCR or qPCR reactions.

Features

High Thermal Stability – Tolerates up to 60°C, ideal for RNA templates with complex secondary structures.

Flexible Primer Options – Compatible with custom primers according to experimental needs.

Broad Compatibility – Efficiently reverse transcribes genes with varying GC content and expression levels.

Strong cDNA Synthesis Capability – Can synthesize cDNA up to 19.8 kb in length.

Components

[Note]: *10× Hifair™ III Super Buffer contains gDNA digester inhibitor and dNTPs.

**HifairTM III RT Enzyme Mix contains RNase inhibitor.

Storage

This product should be stored at -25~-15℃ and protected from light for 18 months.

Application

cDNA Synthesis; Gene Expression Analysis; cDNA Cloning.

Instructions

1. Selection of Reverse Transcription Primers

1) For eukaryotic RNA templates, Oligo (dT)18 is recommended as it pairs with the 3’ poly(A) tail of eukaryotic mRNA, enabling the highest yield of full-length cDNA.

2) For prokaryotic RNA reverse transcription, use Random Primers N6 or gene-specific primers.

3) Random Primers N6 are broadly applicable and suitable for templates including mRNA, rRNA, tRNA, small RNA, and lncRNA.

2. First-Strand cDNA Synthesis Protocol

1) When gDNA Removal is Required

a. gDNA Digestion

In an RNase-free microcentrifuge tube, prepare the following mixture. Mix gently by pipetting and incubate at 42°C for 2 min.

Table 1. gDNA Digestion Reaction System*

[Note]:*For qPCR downstream applications: use 10 pg – 1 μg total RNA or 10 pg – 100 ng mRNA. For low-abundance genes, up to 5 μg total RNA or 500 ng mRNA may be used.

b. Reverse Transcription Reaction Setup (20 μL system)

Add the following components to the previous reaction mixture:

Table 2. Reverse Transcription Reaction System (20 μL example)*

[Note]:*Ensure thorough mixing before use. Avoid vigorous shaking to prevent excessive bubble formation.

1) For qPCR applications, it is recommended to use a 1:1 mixture of Random Primers N6 and Oligo (dT)18.

2) It is recommended to add the 10× Hifair™ III Super Buffer first and mix well before adding the primers to ensure complete inhibition of gDNA digester activity.

3) After assembly, mix gently by pipetting.

c. Standard Reverse Transcription Program

Table 3. Standard Reverse Transcription Program*

[Note]:*Recommended standard protocol.

1) Reverse transcription temperature: 55°C is recommended. For templates with high GC content or complex secondary structures, increase the temperature to 60°C.

2) Reverse transcription time: 15 min is recommended for downstream qPCR; 30–60 min is recommended for downstream PCR.

d. Fast Reverse Transcription Program

Table 4. Fast Reverse Transcription Program*

[Note]:*The fast program is suitable for templates with medium to low GC content (≤55%) or routine templates used in subsequent qPCR experiments.

e. Product Storage

cDNA products can be stored short-term at -20°C. For long-term storage, aliquot and store at -80°C to avoid repeated freeze-thaw cycles.

Documents:

Safety Data Sheet

11139_MSDS_HB251011_EN.PDF

Manuals

11139_Manual_Ver.EN20251011.pdf

Referencs

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