SKU : G3416-10KU
หมวดหมู่ : Biochemicals , 1. Chemical and Reagents , Buffers , Servicebio ,
แบรนด์ : Servicebio
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Product Information
Product Name | Cat. No. | Spec. |
SweScript Reverse Transcriptase II | G3416-10KU | 10 KU |
Product Introduction
Product Description: | SweScript Reverse Transcriptase II is a genetically engineered MMLV reverse transcriptase (RT) based on SweScript Reverse Transcriptase I. Mutations in the RNase H domain of the enzyme avoid degradation of the RNA during first-strand cDNA synthesis, which results in higher yields of full-length cDNA. It has increased thermal stability and higher synthesis efficiency compared to wild-type MMLV RT and SweScript Reverse Transcriptase I. The enzyme is active up between 42-65, providing higher specificity, higher yield of cDNA and more full-length cDNA product up to 5 minutes. |
Applications: | l cDNA synthesis |
Source: | The gene encoding a mutant M-MuLV Reverse Transcriptase is expressed in E. coli. |
Purity: | 95% by SDS-PAGE |
Concentration: | 200 U/μL |
Definition of Activity Unit: | One unit is defined as the amount of enzyme that will incorporate 1 nmol of dTTP into acid-insoluble material in 10 minutes at 37°C using poly(rA)oligo(dT)as template/primer. |
Storage (Dilution) Buffer: | 20 mM Tris-HCl，100 mM KCl，0.1 mM EDTA，1 mM DTT，0.5% Tween 20，0.5% NP-40，50% Glycerol，pH 8.0. |
Storage Conditions: | Store at 20°C up to 12 months. |
Product Contents
Component Number | Component | G3415-10KU |
G3416-1 | SweScript Reverse Transcriptase II | 50 μL |
G3416-2 | 5х Reaction buffer | 500 μL |
Manual | One copy | |
Assay Protocol / Procedures
Protocol of First Strand cDNA Synthesis
1. Thaw components on ice and mix by inverting several times.
2. Mix the following components in a reaction tube:
Component | Volume |
5х Reaction buffer | 4 μL |
dNTP Mix（10 mM each） | 1 μL |
Oligo(dT)18（100 μM） | 1 μL |
or Random Hexamer Primer (100 μM) | or 1 μL |
or Gene Specific Primer (2 μM) | or 1 μL |
RNase inhibitor（40 U/μL） | 1 μL |
SweScript Reverse Transcriptase II | 1 μL |
Total RNA/mRNA | 0.1 ng-5 μg/10 pg-0.5 μg |
Nuclease-free water | To 20 μL |
Total | 20 μL |
Note: For GC rich and complex template, RNA template, primers and nuclease-free water can be premixed. Heat the RNA-primer mix at 65 for 5 minutes, and spin briefly and place promptly on ice.
3. Gently mix and briefly centrifuge.
4. Perform reverse transcription using the recommended thermal conditions below:
Temperature | Time |
25a | 5 min |
50b | 15-30 min |
85 | 5 s |
a: If using Random Hexamer Primer, incubate the combined reaction mixture at 25 for 5 minutes, and then proceed to next step. If using Oligo (dT) 18 Primer or Gene Specific Primer, directly
incubate at 50.
b：For GC rich and complex templates, the reverse transcription temperature can be improved to 65.
Note
1. RNA is easily degraded, Please obey standardized operation to avoid RNase contamination.
2. The reverse transcription products can be stored at -20 for a short period. If long-term storage is required, it is recommended to store at -80 after packing and avoid freeze-thaw cycles.
3. If the template is of eukaryotic origin, it is recommended to select Oligo (dT)18 Primer and pair it with the 3' Poly A tail of eukaryotic mRNA to obtain the highest yield of full-length cDNA.
4. For reverse transcription of prokaryotic RNA, Random Hexamer Primer or Gene Specific Primer should be used.
5. If reverse transcription is followed by qPCR assay, Oligo (dT)18 Primer and Random Hexamer Primer can be mixed to achieve the same cDNA synthesis efficiency in all regions of mRNA, which helps to improve the authenticity and repeatability of quantitative results.
6. For your safety and health, please wear safety glasses, gloves, or protective clot
For Research Use Only!
