MeloScript Reverse Transcriptase

Cat. No.: FP1509103

GRADE & PURITY Suitable for molecular biology, DNase, RNase free, EnzymoPure™, for DNA and RNA applications, 200U/μL

Bioactivity

200 U/μL

Overview

MeloScript Reverse Transcriptase is a novel engineered enzyme developed through in vitro molecular evolution technology. It demonstrates exceptional resistance to common inhibitors (e.g., isopropanol, lysis buffers) present in RNA extracts, enabling reliable performance with suboptimal samples. With superior substrate specificity and catalytic efficiency, it achieves:
✔ High-fidelity reverse transcription of challenging templates (low-abundance, high-GC, or structured RNAs)
✔ Enhanced target capture rate and downstream assay sensitivity

Product Component List

FP1509103	Components	20KU	200KU	2000KU	Storage
FP1509103A	MeloScript Reverse Transcriptase (200U/μL)	100 μL	1.0 mL	10 mL	-20℃
FP1509103B	5×MeloScript buffer	1.0 mL	10×1.0 mL	100 mL	-20℃

Applications

1. One-Step Real-Time RT-PCR

2. First-strand cDNA synthesis

Unit Definition

One unit (U) is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into acid-insoluble material in 10 min at 37°C using poly(rA)·oligo(dT) as template/primer.

Key Guidelines

1. RNase Control

• Maintain a clean workspace with RNase-free consumables (tubes, tips).
• Always wear gloves and masks during handling.

2. Primer Selection
2.1 PCR

• Eukaryotic RNA: Oligo dT (full-length cDNA)
• Prokaryotic RNA/structured regions: Random hexamers
• Target-specific: GSP (Gene-Specific Primer)
2.2 qPCR
• Oligo dT + Random hexamers (balanced coverage)
• GSP for One-/Two-Step RT-qPCR
3. GSP Design Rules

• 3'-end: Prefer G/C; avoid ≥2 consecutive mismatches or hairpins

• Tm: 55–65°C (ΔTm ≤1°C between primers; calculate with Primer Premier 5)

• GC content: 40–60% (uniform base distribution)

• Specificity: Avoid >5 bp self-complementarity or 3' dimerization; verify with NCBI BLAST

Critical Notes

1. Enzyme Handling: Avoid vortexing; keep on ice during use.

2. Enzyme Amount: Excess RT enzyme causes nonspecific PCR products (reduced plateau fluorescence).

Protocol

I. Reaction Setup (25 μL System)

Component	Volume	Final Concentration
5× MeloScript Buffer	5μl	1×
dNTP (25mM each)	0.2μl	0.2mM
Forward Primer (10 uM)	0.5μl	0.2µM
Reverse Primer (10 uM)	0.5μl	0.2µM
Probe (10 uM)	0.25μl	0.1µM
RNase Inhibitor (40 U/μl)	0.25μl	0.4U/μl
MeloScript Reverse Transcriptase (200U/μl)	0.2μl	1.6U/μl
PowerResist Taq Polymerase (5U/μL)	0.5μl	0.1U/μl
Template	5μl	/
Nuclease-free H2O	To 25μl	/
Total	25μl	/

Notes:

• Primer/Probe Optimization: Adjust concentrations (0.1–1 μM primers; 50–250 nM probe)

• Template Dilution: Dilute to 2–5 μL/sample for reproducibility

• Amplicon Length: 80–200 bp (optimal for qPCR)

II. Thermal Cycling

Step	Temperature	Time	Cycles
Reverse Transcription	50℃	15min	1
Initial Denaturation	95℃	2min30s	1
Denaturation	95℃	15s	35-40
Annealing/Extension	55℃	20s	35-40

Optimization Tips:

• RT Temperature: Increase to 55°C for structured/high-GC templates

• Fast Protocols: Reduce RT time to 5 min

• Data Acquisition: Adjust extension time per instrument:

  • ABI 7700/7900HT: ≥30 sec

  • ABI 7000/7300: ≥31 sec

  • ABI 7500: ≥34 sec

Specifications

Product Name

MeloScript Reverse Transcriptase

Grade

DNase, RNase free, for DNA and RNA applications, Suitable for molecular biology, EnzymoPure™

Specifications & Purity

DNase, RNase free,Suitable for molecular biology,EnzymoPure™,for DNA and RNA applications,200U/μL

Bioactivity

200 U/μL

Molecule Type

Enzyme

Storage and Shipping

Concentration

200U/μL

Storage

Store at -20°C,Avoid repeated freezing and thawing

Shipped In

Ice chest + Ice pads

Stability And Storage

Store at -20℃ long term (12 months). Avoid freeze/thaw cycle. Upon receipt, it is recommended to aliquot.

Unit definition

One unit (U) of activity is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into acid-insoluble material in 10 minutes at 37°C, using Poly(rA)·Oligo(dT) as the template/primer.