SweScript RT I First Strand cDNA Synthesis Kit (With gDNA Remover)

Product Information

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. First strand cDNA synthesized with this system can be directly used as a template in subsequent PCR or real-time PCR. 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 has no RNase H activity, which avoids the degradation of RNA in the DNA/RNA hybrid template during the first-strand cDNA synthesis reaction. SweScript RT I has no RNase H activity, avoiding the degradation of RNA in the first-strand cDNA synthesis reaction of DNA/RNA hybrids, thus ensuring the amount and length of first-strand cDNA synthesis. 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.

The gDNA Remover is also provided in this kit, which can quickly remove residual genomic DNA contamination from RNA preps. It improves authenticity and repeatability of the experimental results, and simplify the design of qPCR primers without the need to design primers across exons.

Storage and Shipping Conditions

Ship with Wet ice; Store at -20, valid for 12 months.

Product Contents

a：with RNase Inhibitor.

b：with dNTP Mixture & Mg²+.

Assay Protocol / Procedures

1. Removal of genomic DNA

(1) The RNA template, Nuclease-Free Water was placed on ice to thaw and set aside;.

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

(3) Mix gently and centrifuge briefly；

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

2. First strand cDNA synthesis

(1) Reverse transcription reaction system was prepared (20 μL reaction system recommended) 

Note: For high GC or complex templates, RNA templates, reverse transcription primers, nuclease-free water can be premixed. Heat the RNA-primer mix at 65 for 5 minutes, and Spin briefly and place promptly on ice.

(6) Gently mix and centrifuge briefly.

(7) Perform reverse transcription using the recommended thermal conditions below:

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, directly incubate at 50.

b: For GC rich 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.

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

For Research Use Only!

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