SweScript RT I First Strand cDNA Synthesis Kit
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Product Information
Product Name | Cat. No. | Spec. |
SweScript RT I First Strand cDNA Synthesis Kit | G3330-50 | 50 rxns |
G3330-100 | 100 rxns |
Product Description/Introduction
The SweScript RT I First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from mRNA or total
RNA templates. It contains all components required for 1st strand cDNA synthesis, and provides two kinds of cDNA synthesis primers for your convenience: The Random Hexamer Primer and Oligo (dT)18 Primer. The synthesized single-stranded cDNA products can be directly used for subsequent PCR or qPCR. SweScript RT I (Reverse Transcriptase) is a mutant reverse transcriptase based on M-MLV Reverse Transcriptase and obtained by in vitro evolutionary screening. SweScript RT I is free of RNase H activity, which avoids RNA degradation in the DNA/RNA hybrid template during the first-strand cDNA synthesis reaction, thus ensuring the amount and length of first-strand cDNA synthesized. Compared to the wild-type enzyme, SweScript RT I has dramatically improved thermal stability and synthesis efficiency, and can efficiently synthesize cDNAs in the range of 42-55°C, as well as cDNAs up to 10 kb in length.
Storage and Shipping Conditions
Ship with Wet ice; Store at -20, valid for 12 months.
Product Contents
Component Number | Component | G3330-50 | G3330-100 |
G3330-1 | SweScript RT I Enzyme Mixa | 50 μL | 100 μL |
G3330-2 | 5×Reaction Bufferb | 200 μL | 400 μL |
G3330-3 | Oligo (dT)18 Primer (100 μM) | 50 μL | 100 μL |
G3330-4 | Random Hexamer Primer (100 μM) | 50 μL | 100 μL |
G3330-5 | Nuclease-Free Water | 1 mL | 1 mL |
Manual | One copy | One copy | |
a：with RNase Inhibitor
b：with dNTP Mixture & Mg²+
Assay Protocol / Procedures
First-strand cDNA synthesis steps
1. Reverse transcription reaction system was prepared (20 μL reaction system recommended)
Component | Volume |
5×Reaction Buffer | 4 μL |
Oligo (dT)18 Primer (100 μM) | 1 μL |
or Random Hexamer Primer (100 μM) | or 1 μL |
or Gene Specific Primer (2 μM) | or 1 μL |
SweScript RT I Enzyme Mix | 1 μL |
Total RNA/mRNA* | 0.1 ng-5 μg/10 pg-0.5 μg |
Nuclease-Free Water | Add to 20 μL |
Note: For high GC or complex templates, it is recommended that the RNA template, reverse transcription primers and nucrelease-free water can be mixed in advance and then cooled on ice quickly after being kept at 65 for 5 min. And then you add the other components.
2. Gently mix and centrifuge.
3. Perform reverse transcription using the recommened thermal conditions below:
Temperature | Time |
25a | 5 min |
50b | 5-30 min |
85 | 5 sec |
a: For example, if you usd the Random Hexamer Primer, please incubate at 25 for 5 minutes, then perform the subsequent step. If you choose to use Oligo (dT)18 Primer or Gene Specific Primer, you can directly react at 50.
b: For high GC contnet or complex templates, the reverse transcription temperature can be improved to 55.
Note
1. Please wear disposable gloves when handling 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 repeated 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. Random Hexamer Primer has wide applicability and is suitable for mRNA, rRNA, tRNA, small RNA and lncRNA templates.
6. 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.
For Research Use Only!
