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G3333-50 / 100 SweScript RT II First Strand CDNA Synthesis Kit (With GDNA Remover)

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G3333-50 / 100 SweScript RT II First Strand CDNA Synthesis Kit (With GDNA Remover)

This product utilizes the modified mutant reverse transcriptase to synthesize the first strand of cDNA with total RNA or mRNA as the template for efficient reverse transcription. The kit contains all the reagents needed to synthesize the first strand of cDNA with high quality, and provides two kinds of cDNA synthesis primers for choice: The Random Hexamer Primer and Oligo (dT)18 Primer synthesized single-stranded cDNA products that can be directly used for subsequent PCR or qPCR reactions. SWEScript RT II (Reverse Transcriptase) is a mutant Reverse Transcriptase based on M-MLV Reverse Transcriptase and obtained through in vitro evolutionary screening. SWEScript RT II has no RNase H activity. The RNA degradation in the template of DNA/RNA hybridization during the first strand cDNA synthesis reaction was avoided, so as to ensure the amount and length of the first strand cDNA synthesis. Compared with the wild-type enzyme and the first generation of SweScript RT I, the second generation of SweScript RT II has further improved the thermal stability and cDNA synthesis efficiency. It can efficiently synthesize cDNA in the range of 42-65ºC and complete the reverse transcription reaction as soon as 5 min, especially suitable for the reverse transcription of RNA with complex structure.  

The kit also provides GDNA Remover reagent, which can quickly remove residual genomic DNA contamination from RNA templates before reverse transcription steps, improving the reliability of experimental results, and simplifying the design of qPCR primers without the need for cross-exon primer design.

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Product code Description G3333-50 G3333-100
G3333-1 SweScript RT II Enzyme Mix (a) 50μL 100μL
G3333-2 5x Reaction Buffer (b) 200μL 400μL
G3333-3 Oligo (dT)18 Primer (100uM) 50μL 100μL
G3333-4 Random Hexamer Primer (100uM) 50μL 100μL
G3333-5 gDNA Remover 50μL 100μL
G3333-6 10x gDNA Remover Buffer 50μL 100μL
G3333-7 Nuclease-Free Water 1mL 1mL
  Manual 1pc 1pc

a:with RNase Inhibitor

b:with dNTP Mixture &Mg²+

Technical Specification

Storage and Handling Conditions

Wet ice bag transportation; Stored at -20ºC temperature, valid for 12 months.

Assay Protocol / Procedures

1. Genome removal

(1) The RNA template and Nuclease-free Water were put on ice to melt for later use;

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

Component

Volume

10x gDNA Remover Buffer

1ul

gDNA Remover

1ul

Total RNA/mRNA

0.1 ng-5ug / 10 pg-0.5ug

Nuclease-Free Water

Add to 10ul

(3) Gently mix and centrifuge;

(4) Incubate at 37ºC for 2 min and then put on ice for later use;

2. Synthesis of the first strand cDNA

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

Component

Volume

In the previous step, the genome removed the reaction fluid

10ul

5x Reaction Buffer

4ul

Oligo (dT)18 Primer (100uM)

1ul

or Random Hexamer Primer (100uM)

or 1ul

or Gene Specific Primer (2uM)

or 1ul

SweScript RT II Enzyme Mix

1ul

Nuclease-Free Water

Add to 20ul

Note: For high GC or complex templates, the RNA template, reverse transcription-primers and nuclease-free water can be mixed in advance and then cooled on ice quickly after being kept at 65ºC for 5 min. And then you add the other components.

(2) Gently mix and centrifuge;

(3) Reverse transcription program setting:

Temperature

Time

25ºCa

5 min

55ºCb

5-15 min

85ºC

5 s

a: For example, the Random Hexamer Primer is selected and incubated at 25ºC for 5 min, then the subsequent reaction is carried out. If you choose to use Oligo (dT)18 Primer or Gene Specific Primer, you can directly react at 55ºC.

b: For high GC or complex templates, the reverse transcription temperature can be raised to 65ºC.

Note:

  1. Please wear disposable gloves during operation to avoid RNase contamination.  
  2. The reverse transcription products can be stored at -20ºC for a short time. If long-term storage is needed, it is recommended to store them at -80ºC after packing to 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 make cDNA synthesis efficiency in all regions of mRNA the same, 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.  
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