2. SweScript RT I First Strand cDNA Synthesis Kit (With gDNA Remover), 50 rxns $433,

Product Information

Description

This product uses modified mutant reverse transcriptase to synthesize first-strand cDNA with high efficiency using total RNA or mRNA as template. The kit contains all the reagents needed to synthesize high-quality first-strand cDNA, and provides two kinds of primers for cDNA synthesis: Random Hexamer Primer and Oligo (dT)18 Primer, the single-stranded cDNA product can be directly used for subsequent PCR or qPCR reactions. SweScript RT I (Reverse Transcriptase) is a mutant Reverse Transcriptase obtained through in vitro evolutionary screening based on M-MLV Reverse Transcriptase. SweScript RT I has no RNase H activity. It avoids the degradation of RNA in the DNA/RNA hybrid template during the synthesis of first-strand cDNA, so as to ensure the synthesis amount and length of first-strand cDNA. Compared with the wild-type enzyme, SweScript RT I showed a substantial increase in thermal stability and synthesis efficiency, allowing efficient cDNA synthesis in the range of 42 to 55 ° C, while also producing cDNA up to 10 KB.

gDNA Remover reagent is also provided in this kit, which can quickly remove residual genomic DNA contamination in the RNA template before the reverse transcription step, improve the credibility of the experimental results, and simplify the design of qPCR primers without the need to design primers across exons.

Storage and Handling Conditions

Wet ice transportation; Store at -20℃ for 12 months.

Component

a：include RNase Inhibitor；

b：include dNTP Mixture and Mg²⁺.

Assay Protocol

1.  Removal of genome

(1) The RNA templates, nuclease-free Water, were melted on ice for later use；

(2) Genome removal reaction (10 μL reaction system recommended)：

(3)  Mix gently and centrifuge；

(4)  Incubate at 37 ° C for 2 min and then place on ice for later use;

2.  First strand cDNA synthesis

(1)  Preparation of reverse transcription reaction system (20 μL reaction system is recommended)；

Note: For high GC or complex templates, RNA templates, reverse transcription primers, nuclease-free water can be pre-mixed and held at 65 ° C for 5 min, then quickly cooled on ice. And then you add the other components.

(2) Gently mix and centrifuge;

(3) Reverse transcription program Settings：

a：If Random Hexamer Primer was used, the subsequent reaction was carried out after incubation at 25℃ for 5 min. If Oligo (dT)18 Primer or Gene Specific Primer is used, the reaction can be performed directly at 50℃；

b: For high GC or complex templates, the reverse transcription temperature can be increased to 55 °C.

Note:

1. Wear disposable gloves during operation to avoid RNase contamination.

2. Reverse transcription products can be stored at -20℃ for a short time. If long-term storage is required, it is recommended to store at -80℃ after packaging to avoid repeated freezing and thawing.

3. If the template is of eukaryotic origin, it is recommended to select Oligo (dT)18 Primer, which is paired with the 3′ Poly A tail of eukaryotic mRNA to obtain the highest yield of full-length cDNA.

4. Random Hexamer Primer or Gene Specific Primer should be used for reverse transcription of prokaryotic RNA.

5. Random Hexamer Primer is widely applicable to mRNA, rRNA, tRNA, small RNA, LncRNA and other templates.

6. If reverse transcription is followed by qPCR experiment, Oligo (dT)18 Primer can be mixed with Random Hexamer Primer to make the cDNA synthesis efficiency of each region of mRNA identical, which is helpful to improve the authenticity and repeatability of quantitative results.

7. If the subsequent qPCR primers are designed across exons, the genome removal step can be omitted.

For Research Use Only!

Ver. No.: V1.0-202111