MeloScript Ⅱ Reverse Transcriptase

Cat. No.: FP1508911

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

Overview

  MeloScript Ⅱ Reverse Transcriptase is a next-generation reverse transcriptase obtained through in vitro molecular evolution technology with multiple site-directed mutations of wild-type MMLV reverse transcriptase. Compared to wild-type MMLV reverse transcriptase, MeloScript Ⅱ shows better compatibility with one-step RT-qPCR, significantly reducing inhibition of Taq DNA polymerase while maintaining tolerance to common reaction inhibitors.

  The optimal reaction temperature for MeloScript Ⅱ Reverse Transcriptase is 50-60℃, making it particularly advantageous for processing high-GC templates. At higher temperatures, RNA duplexes unwind more easily, reducing secondary structure formation and thereby improving reverse transcription efficiency and accuracy.

Product Component List

FP1508911	Components	20KU	200KU	2000KU	Storage
FP1508911A	MeloScript Reverse Transcriptase (200U/μL)	100 μL	1.0 mL	10 mL	-20℃
FP1508911B	5×MeloScript buffer	1.0 mL	10×1.0 mL	100 mL	-20℃

Applications

1. Primarily used for one-step Real-Time RT-PCR

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 minutes at 37℃ using poly(rA)·oligo(dT) as template/primer.

Precautions

1. RNase Contamination Control

• Maintain a clean working area

• Wear clean gloves and masks during operation

• Ensure all consumables (centrifuge tubes, pipette tips) are RNase-free

2. Primer Design (Gene-Specific Primers, GSP)

2.1 The 3'-end base should preferably be G or C
2.2 Avoid ≥2 consecutive mismatches in the last 8 bases at the 3'-end
2.3 Avoid hairpin structures at the 3'-end
2.4 Optimal Tm difference between forward and reverse primers should be ≤1℃, with Tm preferably adjusted to 55-65℃ (calculate using Primer Premier 5)
2.5 Additional non-templated sequences should not be included in Tm calculations
2.6 Maintain primer GC content between 40%-60%
2.7 Ensure even distribution of A, G, C, and T bases; avoid regions with extreme GC or AT content
2.8 Avoid >5 bp complementary sequences within primers or between primer pairs; avoid ≥3 bp complementarity at 3'-ends between primers. Verify primer specificity using NCBI BLAST to prevent nonspecific amplification

Additional Notes

1. Enzyme Amount Control

Excessive reverse transcriptase in one-step RT-qPCR reactions may lead to nonspecific PCR products, resulting in decreased fluorescence signals during the plateau phase.

2. Enzyme Handling

• Avoid vigorous vortexing when using MeloScript Ⅱ Reverse Transcriptase

• Keep on ice during use

Protocol

1. Reaction System Preparation

1.1 Thaw and mix all required reagents at room temperature/4℃. Keep on ice or in an ice box.
1.2 Prepare the Real-Time PCR reaction system according to the table below (recommended to prepare on ice):

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 Ⅱ 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	/

1.3 Reaction components can be adjusted according to the following principles:

1. Generally, a final primer concentration of 0.2 μM yields good amplification. If performance is poor, adjust within 0.1-1.0 μM.

2. Probe concentration can be adjusted between 50-250 nM.

3. Due to qPCR's high sensitivity, accurate template addition significantly impacts quantification results. We recommend diluting templates (e.g., to 2-5 μL/sample) before adding to reactions to improve reproducibility.

4. Optimal amplicon length: 80-200 bp.

Reaction Program

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

Notes:

1. For templates with complex secondary structures or high GC content, increasing reverse transcription temperature to 55℃ improves amplification efficiency and sensitivity.

2. For rapid protocols, reverse transcription time can be reduced to 5 min.

3. Adjust extension time according to your Real-Time PCR instrument's minimum data acquisition requirements:

   • ABI 7700 & 7900HT: ≥30 sec

   • ABI 7000 & 7300: ≥31 sec

   • ABI 7500: ≥34 sec

4. Verify instrument compatibility with rapid cycling protocols through preliminary experiments.

Specifications

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.

Bioactivity

200 U/μL

Storage and Shipping

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.

Contents & Storage

FP1508911	Components	20KU	200KU	2000KU	Storage
FP1508911A	MeloScript Ⅱ Reverse Transcriptase (200U/μL)	100 μL	1.0 mL	10 mL	-20℃
FP1508911B	5×MeloScript buffer	1.0 mL	10×1.0 mL	100 mL	-20℃