- cDNA synthesis of up to 15 kb long transcripts
- Template generation for RT-PCR & RT-qPCR
- cDNA synthesis from complex templates
- Thermostable Reverse Transcriptase blended with Ribonuclease Inhibitor for efficient cDNA synthesis
- High yields of full lengths transcripts up to 12-15 kb
- cDNA synthesis from complex templates at up to 55°C
- High sensitivity detection from 1 pg of total RNA template
The HighScriber™ Reverse Transcriptase Mix is a premium tool for the high efficiency reverse transcription of up to 12-15 kb long cDNA. Mix includes HighScriber™ Reverse Transcriptase and Ribonuclease Inhibitor for save, robust cDNA synthesis and ease of use. HighScriber™ Reverse Transcriptase allows for high detection sensitivity from 1 pg of total RNA. The wide reaction temperature range (38°C - 55°C) ensures efficient cDNA synthesis from complex or GC rich templates.
The enzyme uses ssRNA or ssDNA as a template, possesses no detectable Ribonuclease H activity specific to RNA in RNA-DNA hybrids. A highly reduced Ribonuclease H activity allows for transcription of full lengths long transcripts. HighScriber™ Reverse Transcriptase can be used for RACE as it has terminal transferase activity - adds cytosines to 3’ ends of cDNA.
The Ribonuclease inhibitor premixed with the RT ensures RNA protection from ribonuclease degradation.
Supplied 5X ALLin™ HighScriber Buffer includes everything you need for the cDNA synthesis reaction. To minimize pipetting steps it contains MgCl2, dNTPs, enhancers, stabilizers. The only things to add is the template RNA and primer.
- Optimal RT activity is observed at 45-50°C
- RT temperature range 38-55°C
- RT Inactivation at 85°C for 10 min
One unit is defined as the amount of enzyme that will incorporate 1 nmol of dTTP into acid-insoluble material in a total reaction volume of 50 μl in 10 minutes at 37°C using poly (rA) oligo (dT)18 as template.
- RNA is extremely sensitive to degradation by RNases present everywhere. Take care to protect RNA from degradation keep your bench clean, wear gloves, use sterile tubes and filter pipet tips.
- Check the integrity of RNA prior to cDNA synthesis in denaturing agarose gel.
- Include positive control in parallel.
- Thaw and keep reagents on ice. Mix well before use.
- For best results, optimize the template and primer amount.
- Do not exceed the recommended amount of the enzyme mix.
- Perform reaction for 30-50 min, for short transcripts 15-30 min are sufficient.
- Choose optimal reaction temperature in a range of 42-55°C.
- Do not add Ribonuclease Inhibitors and dNTPs, as they are already included in supplied Mix and buffer
Prepare a 20 µl reaction:
|5X ALLin™ HighScriber Reaction Buffer||4 µl (includes dNTPs)|
|Oligo dT primer or
Random primer or
|0.5 µg or
0.2 µg or
|Total RNA or
|1 pg to 5 µg or
1 pg to 0.5 µg
|Water (PCR Water, WAT0110)||to 19 µl|
- Mix gently, avoid bubbles.
- Heat 5 min at 65°C, spin, place on ice for 1 min.
- Incubate 2 min at 42°C for Oligo dT and for Specific primer
or 10 min at 25°C for Random primer to anneal.
- Add 1 µl of HighScriber™ Reverse Transcriptase Mix, 20X (Includes RT and Ribonuclease Inhibitor)
- Incubate 30-50 min at 50°C to synthesize cDNA.
- Inactivate at 85°C for 10 min.
- Store reactions at -20°C or on ice for an immediate use.
- Use 2-5 µl of this reaction mix per 50 µl PCR reaction.
- Use 2 µl of this reaction mix per 20 µl qPCR reaction.
2 x 25 µl - HighScriber™ Reverse Transcriptase Mix, 20X
2 x 0.2 ml - 5X ALLin™ HighScriber Buffer
Enzyme Mix contains HighScriber™ Reverse Transcriptase at 200 u/µl concentration, Ribonuclease Inhibitor and glycerol. 5X ALLin™ HighScriber Buffer contains MgCl2, dNTPs, enhancers, stabilizers..
10 x 25 µl - HighScriber™ Reverse Transcriptase Mix, 20X
10 x 0.2 ml - 5X ALLin™ HighScriber Buffer