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SweScript RT I First Strand CDNA Synthesis Kit

  • G3330-50

Product Description

Product introduction:

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 I (Reverse Transcriptase) is a mutant Reverse Transcriptase based on M-MLV Reverse Transcriptase and obtained through in vitro evolutionary screening. SWEScript RT I 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, the thermal stability and synthesis efficiency of SweScript RT I are significantly improved, and cDNA can be synthesized efficiently in the 42-55℃ range, as well as cDNA as long as 10kb .

 

Package Contents:

Cat. No.

Product description

Volume

G3330

SweScript RT I First Strand cDNA Synthesis Kit

50rxns/100rxns

 

Components:

Component Number

Component

G3330-50

G3330-100

G3330-1

SweScript RT I Enzyme Mixa

50ul

100ul

G3330-2

5×Reaction Bufferb

200ul

400ul

G3330-3

Oligo (dT)18 Primer (100uM)

50ul

100ul

G3330-4

Random Hexamer Primer (100uM)

50ul

100ul

G3330-5

Nuclease-Free Water

1mL

1mL

Manual

1pc

1pc

a:with RNase Inhibitor

b:with dNTP Mixture & Mg²+

 

Storage condition:

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

 

Usage:

The first step for cDNA synthesis

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

Component

Volume

5×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 I Enzyme Mix

1ul

Total RNA/mRNA*

0.1 ng-5ug/10 pg-0.5ug

Nuclease-Free Water

Add to 20ul

Note: For high GC or complex templates, the RNA template, reverse transcription-primers and nucrenase-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 reaction components.

2. Gently mix and centrifuge;

3. Reverse transcription program setting:

 

Temperature

Time

25℃a

5 min

50℃b

15-30 min

85℃

5 s

a: For example, the Random Hexamer Primer is selected and incubated at 25℃ 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 conduct a reaction at 50℃;

B: For high GC or complex templates, the reverse transcription temperature can be raised to 55℃.

 

Notes:

1. Please wear disposable gloves during operation to avoid RNase contamination.  

2. The reverse transcription products can be stored at -20℃for a short time. If long-term storage is needed, it is recommended to store them at -80℃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.  

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