Reverse Transcriptase First Strand cDNA Synthesis Kit
Product Components
| Component | Component Number | Size (100 RXN) |
|---|---|---|
| Molzyme II Enzyme Mix (10X) | MZ21451 | 200 µL |
| Molzyme II Reaction Mix (2X) | MZ21450 | 1 mL |
| Oligo(dT)23VN* (50 µM)** | MZ20115 | 200 µL |
| Random Primer Mix (60 µM)** | MZ20116 | 200 µL |
| dNTPs (10 mM each) | MZ20120 | 100 µL |
| Nuclease-free H₂O | MZ20214 | 1.25 mL |
* V = A, G, or C; N = A, G, C, or T.
** Contains 1 mM dNTPs.
Product Description
Molzyme™ Reverse Transcriptase First Strand cDNA Synthesis Kit features two optimized mixes: Molzyme™ II Enzyme Mix and Molzyme™ II Reaction Mix. The enzyme mix combines Molzyme™ II Reverse Transcriptase and RNase Inhibitor, while the reaction mix contains an optimized buffer system.
Molzyme™ II Reverse Transcriptase is a recombinant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability. It can synthesize first-strand cDNA at higher temperatures than wild-type M-MuLV. The enzyme is active up to 48°C, providing higher specificity and improved cDNA yield.
The kit also includes two optimized primers for reverse transcription and nuclease-free water. An anchored Oligo-dT primer [d(T)23VN] forces the primer to anneal at the beginning of the poly(A) tail. The optimized Random Primer Mix provides random and consistent priming sites across the entire RNA template, including both mRNA and non-polyadenylated RNA.
The first-strand cDNA generated can be up to 10 kb in length.
Quality Controls
The performance of the Reverse Transcriptase First Strand cDNA Synthesis Kit is tested in a reverse transcription reaction using Jurkat total RNA with primer d(T)23VN. The length of the synthesized cDNA is verified by detection of a 9.2 kb amplicon of the fibrillin gene.
First Strand cDNA Synthesis Reaction
Denaturation of RNA and primer at 65–70°C for 5 minutes can remove secondary structures that may impede long cDNA synthesis. However, this step can be omitted in many cases (unpublished results).
We recommend incubation at 42°C for one hour for maximum yield and length. However, many targets can be detected after a much shorter incubation time. For example, 10 minutes incubation can be used for up to 5 kb cDNA synthesis.
Choice of Primers for Reverse Transcription
Oligo-dT primer is preferred for most applications because it ensures that all cDNA copies terminate at the 3′ end of the mRNA and produces the longest contiguous cDNA. An anchored oligo-dT primer [d(T)23VN] forces the primer to anneal to the start of the polyA tail, thereby preventing priming at internal sites in the polyA tail (1).
The Random Primer Mix is an optimized mix of hexamer and dNTP. It provides random priming sites covering the entire RNA templates, including both mRNAs and non-polyadenylated RNAs (such as ribosomal RNAs). The Random Primer Mix yields shorter cDNAs on average and can be used for the detection of multiple short RT-PCR products. Random Primer Mix offers good performance in a wide range of RNA templates.
When a gene-specific primer is used in a cDNA synthesis reaction, the cDNA product can be used only for amplification of that transcript. This priming method gives good results when the amount of RNA is limiting (below 10 ng) and only one particular cDNA is desired.
- Recommended primer concentration:
| Primer Type | Oligo-dT23VN | Random Primer Mix | Gene-Specific Primer |
|---|---|---|---|
| Final Concentration | 5 µM | 6 µM | 0.1–1 µM |
First Strand cDNA Synthesis Protocols
Thaw kit components on ice and mix by inverting several times.
Easy Protocol
Mix the following components and incubate at 42°C for 1 hour. If Random Primer Mix is used, an incubation step at 25°C for 5 minutes is recommended before the 42°C incubation.
| Component | 1X RXN |
|---|---|
| Template RNA | up to 1 µg |
| d(T)23VN (50 µM) | 2 µL |
| 10 mM dNTPs | 1 µL |
| Molzyme II Reaction Mix (2X) | 10 µL |
| Molzyme II Enzyme Mix (10X) | 2 µL |
| Nuclease-free H₂O | to 20 µL |
Inactivate the enzyme at 80°C for 5 minutes. For downstream PCR application, the volume of cDNA product should not exceed 1/10 of the PCR reaction volume.
Standard Protocol (With RNA Denaturation)
If denaturation of template RNA is desired, use the following protocol.
Mix RNA sample and primer d(T)23VN in a sterile RNase-free microfuge tube:
| Component | 1X RXN |
|---|---|
| Total RNA | 1–5 µL (up to 1 µg) |
| d(T)23VN (50 µM) | 2 µL |
| 10 mM dNTPs | 1 µL |
| Nuclease-free H₂O | to 8 µL |
Denature sample RNA/d(T)23VN for 5 minutes at 65°C. Spin briefly and place promptly on ice.
Add the following components:
| Component | 1X RXN |
|---|---|
| Molzyme II Reaction Mix (2X) | 10 µL |
| Molzyme II Enzyme Mix (10X) | 2 µL |
Incubate the 20 µL cDNA synthesis reaction at 42°C for one hour. If Random Primer Mix is used, an incubation step at 25°C for 5 minutes is recommended before the 42°C incubation.
Inactivate the enzyme at 80°C for 5 minutes. The cDNA product should be stored at −20°C. In general, the volume of cDNA product should not exceed 1/10 of the PCR reaction volume.
No-RT Negative Control Reaction
Mix the following components and incubate at 42°C for 1 hour:
| Component | 1X RXN |
|---|---|
| Template RNA | up to 1 µg |
| d(T)23VN (50 µM) | 2 µL |
| Molzyme II Reaction Mix (2X) | 10 µL |
| 10 mM dNTPs | 1 µL |
| Nuclease-free H₂O | to 20 µL |