SweScript Reverse Transcriptase I
Cat. No.: SB-G3415-10KU
Size: 10KU
Description
This product, SweScript Reverse Transcriptase I, is a reverse transcriptase mutant obtained through in vitro transformation and screening based on wild-type M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase. It has no RNase H activity, avoiding the degradation of RNA in the DNA/RNA hybrid template in the first-strand cDNA synthesis reaction, thereby ensuring the amount and length of the first-strand cDNA synthesis; compared with the wild-type enzyme, SweScript Reverse Transcriptase I has a higher heat The stability and synthesis efficiency are greatly improved, and cDNA can be efficiently synthesized in the range of 42-55°C, and cDNA up to 10 kb can be synthesized.
- Source : derived from M-MLV (Moloney Murine Leukemia Virus), recombinantly expressed by Escherichia coli
- Definition of enzyme activity: at 37°C, within 10 minutes, using Poly(rA) Oligo (dT) as template/primer, the amount of enzyme required to incorporate 1 nmol of dTTP as an acid-insoluble substance is defined as one enzyme activity unit (U)
- Purity and concentration: SDS-PAGE detection purity ≥ 95%; endogenous nucleic acid residue < 1 pg/μL (qPCR detection); 200 U/μL.
- Enzyme Storage Buffer: 20mM Tris-HCl, 100mM KCl, 0.1mM EDTA, 1mM DTT, 0.5% Tween 20, 0.5% NP-40, 50% Glycerol, pH 8.0;
Storage and transportation
Transport in wet ice packs; store at -20°C, valid for 12 months.
Product Contents
| Component Number |
Component |
G3415 |
| G3415-1 |
SweScript Reverse Transcriptase I |
50 μL |
| G3415-2 |
5x Reaction buffer |
500 μL |
| Product Manual |
1 copy |
Steps
First-strand cDNA synthesis steps
1. Configure the reverse transcription reaction system (20 μL reaction system is recommended):
| Component |
Volume |
| Total RNA/mRNA |
0.1 ng-5 μg/10 pg-0.5 μg |
| SweScript Reverse Transcriptase I |
1 μL |
| RNase inhibitor (40 U/μL) |
1 μL |
| or Gene Specific Primer (2 μM) |
or 1 μL |
| or Random Hexamer Primer (100 μM) |
or 1 μL |
| Oligo(dT)18 (100 μM) |
1 μL |
| dNTP Mix (10 mM each) |
1 μL |
| 5x Reaction Buffer |
4 μL |
| Nuclease Free Water |
To 20 μL |
Note: For high GC or complex templates, pre-mix RNA templates, reverse transcription primers, nuclease-free water and incubate at 65°C for 5 minutes, then quickly cool on ice. Then the other reaction components are added.
2. Gently mix and centrifuge;
3. Reverse transcription program settings:
| temperature |
Time |
| 25℃a |
5 minutes |
| 50℃b |
15-30 minutes |
| 85°C |
5 seconds |
a: If you choose to use Random Hexamer Primer, incubate at 25°C for 5 minutes before performing the subsequent reaction; if you choose to use Oligo (dT) 18 Primer or Gene Specific Primer, you can directly perform the reaction at 50°C;
b: For high GC or complex templates, the reverse transcription temperature can be increased to 55°C.
Precautions
1. RNA is easy to be degraded, standardize the operation to avoid RNase contamination;
2. The reverse transcription product can be stored at -20°C for short-term storage. If it needs to be stored for a long time, it is recommended to store it at -80°C after aliquoting to avoid repeated freezing and thawing;
3. If the template is of eukaryotic origin, it is recommended to choose Oligo (dT) 18 Primer, which can be paired with the 3' Poly A tail of eukaryotic mRNA to obtain the highest yield of full-length cDNA;
4. For prokaryotic RNA reverse transcription, please use Random Hexamer Primer or Gene Specific Primer;
5. If reverse transcription is followed by qPCR experiments, Oligo (dT) 18 Primer and Random Hexamer Primer can be used in combination, which can make the cDNA synthesis efficiency of each region of mRNA the same, and help improve the authenticity and repeatability of quantitative results;
6. For your safety and health, please wear a lab coat and disposable gloves for operation.
