HighYield T7 RNA Crosslinking Kit (4-Thio-UTP)

HighYield T7 RNA Crosslinking Kit (4-Thio-UTP) is designed to produce randomly 4-Thio-modified RNA probes via in vitro transcription. Such probes are ideally suited for the detection of RNA-Protein interactions by photocrosslinking. The photoreactive Thio group triggers the formation of a covalent bond between the RNA and closely interacting proteins upon UV irradiation. 4-Thio-UTP is efficiently incorporated into RNA as substitute for its natural counterpart UTP using an optimized reaction buffer and T7 RNA Labeling Polymerase Mix. 80% 4-Thio-UTP substitution typically results in an optimal balance between reaction and labeling efficiency. Individual optimization of 4-Thio-UTP/UTP ratio however, can easily be achieved with the single nucleotide format. The result-ing 4-Thio-modified RNA probe can be used for photocrosslinking experiments using a 365 nm UV light. The kit contains sufficient reagents for 15 labeling reactions of 20 μl each (100% 4-Thio-UTP substitution, 7.5 mM ATP, 7.5 mM GTP, 7.5 mM CTP, 7.5 mM 4-Thio-UTP) . A 20 μl reaction with 100% substitution yields about 10 μg RNA, with 80% substitution about 50 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) .