HighYield T7 P&L RNA NMR Kit (2F-ATP)

HighYield T7 P&L RNA NMR Kit (2F-ATP) is designed to produce large amounts of 2-Fluoro-modified RNA via in vitro transcription with T7 RNA polymerase. The resulting internally modified (m) RNA can subsequently be used for structure determination vianuclear magnetic resonance (NMR) spectroscopy. 2F-ATP is incorporated instead of its natural counterpart in a non-perturbing way (intact base pairing properties) . 2-Fluoro-modification simultaneously functions as a sensitive NMR reporter group (larger chemical shift dispersion than1H) . The modified T7 RNA polymerase (T7 P&L RNA polymerase) with proline 266 replaced by leucine (P266L) has been associated with increased 5' homogeneity of transcripts especially when synthesized from A-initiating phi2.5 promoter. The kit contains sufficient reagents for 15 reactions (S-Pack) or 50 reactions (L-Pack) á 20 μl (7.5 mM GTP, 7.5 mM 2-Fluoro-ATP, 7.5 mM UTP, 7.5 mM CTP) . An individual optimization of 2-Fluoro-ATP concentration can easily be achieved with the single nucleotide format. A 20 μl reaction yields about 80 μg RNA after 30 min incubation (1 μg T7 control template, 1.4 kb RNA transcript) . Yields may however vary depending on the template (promotor design, sequence length, secondary structure formation) .