thermostable cdna synthesis supermix (+gdna...
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Order & InquiryTel: +49-89-46148500 Fax: +49-89-461485022E-mail: [email protected]
ThermoStable cDNA Synthesis SuperMix (+gDNA Remover)
1. General Information
Notice！● This product is designed for use in both RT-PCR and RT-qPCR.● This product achieves simultaneous cDNA synthesis and genomic DNA removal.
ThermoStable cDNA Synthesis SuperMix (+gDNA Remover) is engineered to have high thermal stability (at 65°C) for efficient transcription of RNA regions with high secondary structure. It functions over a broad range of reaction temperatures (42-65°C), and achieves simultaneous genomic DNA (gDNA) removal and cDNA synthesis. This product generates high yields of cDNA, and is able to synthesize very long RNA transcripts up to 20 kb. The cDNA products are suitable for downstream PCR, qPCR and RNA-Sequencing applications.
2. Contents
Stored at -20°C for up to one year. It is recommended to pre-aliquot the mix into small batches for frequent usage. Product quality is guaranteed under proper storage conditions.
3. Storage and Stability
The following protocol has been optimized for generating first-strand cDNA for use in both RT-PCR and qRT-PCR.
4. Protocol
Component B24201
ThermoStable RT/RI Enzyme Mix
gDNA Remover
2 × TS ThermoStable Reaction Mix
Random Primer (N9) (0.1 µg/µL)
Anchored Oligo (dT)18 Primer (0.5 µg/µL)
RNase-free Water
50 µL
50 µL
500 µL
50 µL
50 µL
500 µL
B24202
100 µL
100 µL
1 mL
100 µL
100 µL
1 mL
1. Combine the following components in a tube on ice.
Component Volume
Total RNA/mRNA
2 × TS ThermoStable Reaction Mix
ThermoStable RT/RI Enzyme Mix
gDNA Remover
RNase-free Water
50 ng-5 µg/5-500 ng
10 µL
1 µL
1 µL
Up to 20 µL
1 µL/1 µL/2 pmolAnchored Oligo (dT)18 Primer (0.5 µg/µL)/Random Primer (N9) (0.1 µg/µL)/ GSP (gene specific primer)
Note: for higher efficiency of reverse transcription, first mix RNA, primers and RNase-free Water together, followed by incubation at 65°C for 5 minutes. Then cool on ice for 2 minutes, until tube contents reach room temperature, prior to adding other components into the tube.
2. Gently mix tube contents and incubate at
Incubate tube at 85°C for 5 seconds to inactivate ThermoStable Reverse Transcriptase and gDNA Remover and then chill on ice.
4. Store at -20°C until use.
65°C for 30 minutes (for anchored Oligo (dT)18 Primer or GSP)25°C for 10 minutes, and then at 65°C for 30 minutes (for Random Primer (N9))
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3.
5. Important NotesGently invert the tube upside down several times before use. Do NOT vortex. Brief centrifugation prior to use is recommended.
1.
High-quality RNA templates are recommended to ensure successful cDNA synthesis.
2.
Q1:
A1:
3. For RNA templates of complex structure or to achieve higher synthesis efficiency for standard RNA templates, extend incubation of template and primers as described.
6. TroubleshootingMy mRNA has a high-GC content. Which reverse transcriptase do you recommend using?
We would recommend using our ThermoStable cDNA Synthesis SuperMix, which provides the highest thermostability of any RT on the market to address your challenging template (high-GC content or extensive secondary structure).