Foreasy New Generation Of M-MLV Reverse Transcriptase RT Enzyme Foreasy
Foreasy M-MLV Reverse Transcriptase
Foreasy New Generation Of M-MLV Reverse Transcriptase RT Enzyme Foreasy For Sale
Product Introduction
Foreasy M-MLV Reverse Transcriptase is a new reverse transcriptase expressed in E. coli engineered bacteria using genetic recombination technology. It is a recombinant DNA polymerase that synthesizes a complementary DNA strand from single-stranded RNA, DNA, or an RNA:DNA hybrid. It has no RNase H activity, strong stability, strong RNA affinity, and high detection sensitivity.
Product components:
| Component | IM-02011 | IM-02012 | IM-02013 |
|
Foreasy Reverse Transcriptase (200 U/μL) |
200 KU (1 mL) | 2000 KU (10 mL) | 5000 KU (25 mL) |
|
5× Foreasy RT Buffer |
1 mL ×5 | 10 mL ×5 | 10 mL ×12 |
Type
| Cat No. | Type | |
| IM-02011 | 200 KU | 200 U/μL |
| IM-02012 | 2,000 KU | 200 U/μL |
| IM-02013 | 5,000 KU | 200 U/μL |
Storage
Store at -20±5℃ for 2 years or at -80℃ for long-term storage.
Application
u Synthesis of the first strand of cDNA for RT-PCR and real-time RT-PCR
u cDNA synthesis for cloning and expression.
u Generation of labeled cDNA probes for microarrays.
u Primer extension RNA analysis
Unit definition
One unit of M-MLV RT is the amount of enzyme required to incorporate 1 nmole of [ 3H] dTTP in 10 min at 37°C using poly(A)oligo(dT)25 as template-primer.
Unit reaction conditions
50 mM Tris-HCl (pH 8.3), 40 mM KCl, 6 mM MgCl2, 1 mM DTT, 0.5 mM [3H]dTTP, 0.1 mM poly(A), 0.1 mM oligo(dT)25, 0.1 mg/mL BSA, and enzyme in 50 µL for 10 min at 37°C.
Operation :
synthesize the first strand cDNA (20 μL reaction system)
1. RT system structure (the following steps should be performed on ice).
1.1 After defrosting each compoment, add it to the reaction tube in the order of following Table 1
Table 1:
|
RT system add content
|
Volume |
Final concentration
|
| Template RNA | X µL | Total RNA:<5 μg / mRNA:<0.5 μg |
| Random Primer | 0.5 µg | 100 pmol |
| Or Oligo(dT)18 Primer | 0.2 µg | 100 pmol |
| Or Specific Primer | 15-20 pmol | 15-20 pmol |
| RNase-Free ddH2O | to 12.5 µL | |
1.2 After the system is prepared, mix gently and centrifuge briefly, then perform RT reaction according to the below in Table 2
Table 2
| Step | Temperature | Time | Content |
| 1 | 65℃ | 5 min | Denaturation |
| Cool quickly on ice after finishing the reaction | |||
- Adding the Reactant in the order as the following table 3 after cool on the ice
Table:3:
|
RT system add content
|
Volume |
Final concentration
|
| 5× Foreasy RT Buffer | 4 µL | 1× |
| Foreasy RNase Inhibitor (40 U/µL) | 0.5 µL | 1 U/μL |
| dNTP Mix (10 mM each) | 2 µL | 1 mM |
| Foreasy Reverse Transcriptase (200 U/µL) | 1 µL | 10 U/μL |
1.4 After the system prepared, mix gently and centrifuge briefly, then perform RT reaction conditions in following Table 4
Table 4:
| Step | Temperature | Time | Content |
| 1 | 42℃ | 50 min | cDNAsynthesis |
| 2 | 70℃ | 10min | Inactivated reverse transcriptase |
| 3 | 4℃ | N/A | Place it at 4°C for later use or store at -20°C after reaction |
Note: When using Random Primer, the reaction should be preheated at 25°C for 10 minutes before the first step reaction.
The above procedure is for reference only, and the actual reaction conditions vary depending on the structure of the template, primers, etc.
After the reaction is completed, the reaction product is placed on ice for subsequent experiments directly; for long-term storage, please store it at -20°C or -80°C.
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