Optimized buffer for reverse transcription
For general laboratory use.
Shipping: shipped at ambient temperature
Storage Conditions: store at 4 °C or -20 °C
Shelf Life: 12 months
Concentration: 5 x conc.
SCRIPT RT Buffer complete (5 x conc.) is optimized for use with SCRIPT Reverse Transcriptase (#PCR-425) in reverse transcription applications. The enzyme / buffer combination is recommended for synthesis of cDNA from 100 bp up to 10 kb length.
SCRIPT RT Buffer complete
250 mM Tris-HCl (pH 8.5), 500 mM KCl, 25 mM MgCl2, stabilizers
Recommended protocol for cDNA synthesis:
Sample denaturation prior to the assay set-up is only recommended for RNA targets that exhibit a high degree of secondary structure, for self- or cross-complementary primers and for initial experiments with new targets. In this case incubate the mixture of RNA and primers at 65-70 °C for 5 min and place it at room temperature (if using a specific primer) or on ice (if using oligo-dT or random primer).
Add the following components to a nuclease-free microtube. Pipett on ice and mix the components by pipetting gently up and down. In general, water, RNA and primers should be mixed together before the remaining components are added.
|SCRIPT RT Buffer complete||1 x|
|dNTP Mix||500 μM each dNTP|
|SCRIPT Reverse Transcriptase 1)||100-200 units/assay|
|RNase Inhibitor 2)||20 units/assay|
10-20 pmol (50-100 ng)
50 pmol (300 ng)
50 pmol (100 ng)
|PCR-grade Water||fill up to final volume|
Use 10 pg - 5 μg of total RNA or 10 pg-500 ng of mRNA per assay.
1) 100 units (0.5 μl) of enzyme is recommended for standard assays but increasing the amount of enzyme to 200 units (1 μl) per assay may show even higher transcription yields under selected assay conditions.
2) Addition of 20-40 units RNase inhibitor per assay is recommended and may be essential when working with low amounts of starting RNA.
First-strand cDNA synthesis
Incubate the reaction mix at 50 °C for 30-60 min if using gene-specific primers. If using oligo-dT or random primers incubate at 42°C for
10 min followed by incubation at 50°C for 30-60 min.
Please note: The optimal time depends on the length of cDNA. Incubation of 60 min is recommended for cDNA fragments of more than 2,000 bp length. The optimal temperature depends on the structural features of the RNA. Increase the temperature to 55 °C for difficult templates with high secondary structure. Note that optimal reaction time and temperature should be adjusted for each particular RNA.
Optional: Heat inactivation
Heat the mixture to 70°C for 10 min to inactivate the Reverse Transcriptase.
Optional: RNA removal
Add 2 units DNase-free RNase and incubate at 37°C for 20 min.
The cDNA can now be used as template in PCR or be stored at -20°C. Apply 2-5 μl of the RT assay for further amplification in PCR.
However, some specific DNA applications may require prior inactivation of the remaining RTase or the enzymatic removal of RNA.