Hypersensitive M-MLV Reverse Transcriptase

Composition

Storage

It can be store at -20℃ for two years.

Introduction

Hypersensitive M-MLV Reverse Transcriptase is the third generation M-MLV reverse transcriptase obtained by gene modification and recombination technology. Compared with wild-type M-MLV reverse enzyme, this enzyme removes RNase H activity and significantly improves reverse transcription speed and thermal stability (maximum tolerance temperature is 60℃), and enhances tolerance to the complex secondary structure of RNA. Highly sensitive reverse transcriptase has strong anti-interference and very high reverse transcriptase efficiency for low-copy RNA template, and is the preferred reverse transcriptase for RT-PCR nucleic acid detection kit.

Application

1. cDNA library construction.

2. RT-PCR and Real Time RT-PCR.

3. Primer extension.

4. RNA sequencing.

5. One-step RT-PCR

Unit Definition

Product concentration of 200 U/µl. Definition of activity unit: Using Poly (A) as template and Oligo (dT) as primer, the amount of enzyme required to catalyze the incorporation of 1 nmol dTTP within 10 min at 37℃ was defined as 1 activity unit (U).

Purity

The purity of the product is more than 90% (by Coomassie brilliant blue staining SDS-PAGE). The product is free of endonucrenase, exonucrenase and RNase contamination.

Equipment and Reagents to Be Supplied by User

1.        oligo(dT) 12- 18 (25 μM) or random primers (25 μM) or gene specific primers (1 μM)

2.        dNTPs (10 mM each)

3.        RNase Inhibitor may be required

4.        RNase-free microcentrifuge tube

5.        Pipette and tips (To avoid RNase contamination, RNase-free tip with filter must be selected)

6.        Disposable gloves, facemasks and other protective items

7.        Water bath

8.     A laboratory without RNase: Because RNase is present in saliva and skin, please wear latex gloves and a mask during the whole experiments.

Protocol

1.     Add the following reagents to a RNase-free microcentrifuge tube

1)     2 µl oligo(dT)12-18 (10 μM) or 2 µl random primer (10 μM) or 2 pmol gene-specific primer.

2)     0.5-5 μg total RNA or 50-500 ng mRNA;

* When total RNA is less than 0.5 μg (such as viral RNA reverse transcription), the amount of M-MLV reverse transcriptase should be reduced to 0.05-0.5 µl, otherwise it may lead to subsequent PCR amplification to produce non-specific amplification products.

* When total RNA is less than 0.5 μg, it was recommended to add 1 μl RNase Inhibitor

* If the RNA template needs to be incubated at 70℃ for 5 min to destroy the secondary structure, the addition of RNase Inhibitor should not be omitted.

3)     1 µl dNTPs (10 mM each).

4)     Add RNase-free Water to 15 µl.

* If the RNA template is GC-rich or has a complex secondary structure, add the following steps: Incubated at 70℃ for 5 min to destroy the RNA secondary structure, then quickly place on ice to prevent the secondary structure from re-forming, then spin down.

2.     Add the reagent according to the table below:

* When total RNA is less than 1 μg, the amount of M -MLV reverse transcriptase should be reduced to 0.05-0.5 µl.

3.     Mix gently and incubate at 25℃ for 10 min with random primers as primer.

4.     Incubate at 42℃ for 60 min.

5.     Incubate at 95℃ for 5 min, then cool on ice or store below -20℃ for later use.

6.     Add RNase-free Water to 50 µl and take 2-5 µl for PCR amplification.