RNA Synthesis of Cloned Insert Transcripts
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RNA Synthesis Protocol
- Linearize the purified plasmid containing your cloned insert downstream of the insertion site utilizing any of the restriction enzyme sites flanking the insertion site after confirming your insert does not contain any internal sites for your chosen restriction enzyme. We recommend restriction digests leaving blunt or 5´ overhang ends.
- Purify and quantitate your DNA by use of Nanodrop, Qubit or UV absorption approaches.
- Assemble the RNA synthesis reaction at room temperature in the following order:
COMPONENTS AMOUNT FINAL CONC. Nuclease-free Water X µl RNAPol Reaction Buffer (10X) 2 µl 1X Ribonucleotide Solution Mix (25 mM each) 3.2 µl 4 mM each Template DNA X µl 0.2-1 µg MgCl2 (100 mM) 2.8 µl additional 14 mM* RNase Inhibitor, Murine or Human Placenta (40 units/µl) 0.5 µl 1 unit/µl final Fresh DTT (100 mM) (optional) 1 µl 5 mM final T7 RNA Polymerase (50 units/µl) or SP6 RNA Polymerase (20 units/µl) 2 µl 100 units (T7) or 40 units (SP6) Total Volume 20 µl
*1X reaction buffer contributes 6 mM for a final concentration of 20 mM.
- Incubate at 37°C for 1 hour. For shorter (< 300 nt) transcripts incubate at 37°C for 2-4 hours.