Warm Start RTL Reverse Transcriptase（Glycerol free）

WS RTL Reverse Transcriptase 2.0 (WS RTL 2.0) is an aptamer-modified and RNA template dependent DNA polymerase that inhibits its reverse transcription activity below 40°C. WS RTL 2.0 lacks 3′ →5′ exonuclease activity and has RNase H activity. The enzyme can use RNA as a template to synthesize a complementary DNA strand, which can be applied to first-strand cDNA synthesis. Because of its high activity between 50-65°C, it is particularly suitable for RT-LAMP (Loop-Mediated Isothermal Amplification). Since the polymerase activity of WS RTL 2.0 is inhibited below 40°C, the non-specific amplification caused by mismatching of primers during the preparation of the reaction system can be greatly reduced, solving the problems of primer-dimer formation before the reaction, improving the consistency and specificity of amplification. WS RTL 2.0 supports high-throughput applications and operation at room temperature. WS RTL reverse transcriptase 2.0 (Glycerol free) can be used to prepare lyophilized preparations, lyophilizable RT-LAMP reagents, etc.

Components

Storage Condition

Transportation under 0°C and be stored at -25°C~ -15°C. Avoid repeated freezing and thawing, and valid for 18 months.

Unit definition

One unit incorporates 1 nmol of dTTP into acid-precipitable material in 20 minutes at 50 °C using poly(A)•oligo(dT) as template-primer.

Quality Control

Endonuclease Activity: Incubation of 15 U of enzyme with 1 μg λ DNA for 16 hours at 37°C resulted in no detectable degradation of the DNA as determined by gel electrophoresis.

Exonuclease Activity: Incubation of 15 U of enzyme with 1 μg λ-Hind III digest DNA for 4 hours at 37°C resulted in no detectable degradation of the DNA as determined by gel electrophoresis.

Nickase Activity: Incubation of 15 U of enzyme with 1μg pBR322 for 4 hours at 37°C resulted  in no detectable degradation of the DNA as determined by gel electrophoresis.

RNase Activity: Incubation of 15 U of enzyme with 0.48μg MS2 RNA for 4 hours at 37 ℃ resulted in no detectable degradation of the RNA as determined by gel electrophoresis.

E.coli DNA: 15 U of enzyme is detected by TaqMan qPCR. The E.coli DNA is <1 copy.

Reaction Setup

cDNA Synthesis Protocol

Notes:

1) The recommended dosage of Total RNA is 1ng~1μg

2) The recommended dosage of mRNA was 50ng~100ng

Reaction process：

Notes:

1) a .If Random Primer Mix is used, an incubation step at 25°C.

2) b. If target primer mix is used, an incubation step at 55°C for 10~30mins. Longer reverse  transcription time helps to obtain longer cDNA (>5 kb).

RT-LAMP Protocol

Notes:Mix by vortexing and centrifuge briefly. Then keep at 65°C for 1 hour.

Note: The two buffers are interoperable and have the same composition.

Notes:

1. This product may appear white precipitate after stored at -20 °C. It is a normal observation. Please keep it in ice for 10 min to make the precipitate get dissolved and mix well before use.
2. The cDNA product can be stored at -20°C or -80°C or used immediately for PCR reaction.
3. To prevent RNase contamination, please keep the working area clean, and wear cleaned gloves and masks during operation. The centrifugal tube, suction and other consumables used in the experiment should be guaranteed to be RNase-free.