Spermidine → Biotin

Participants

Reaction Steps

Each transcription reaction with T7 or T3 RNA polymerase is carried out in an Eppendorf tube with the following conditions: DEPC H20 q.s. 20 μl; 40 mM Tris-HCl pH 7.5; 6 mM MgCl2; 2 mM spermidine; 5 mM NaCl; 10 mM DTT; 100 μg/ml bovine serum albumin (BSA) (fraction V, Sigma); 500 μM CTP, ATP, GTP; variable amounts of UTP, biotin-UTP and DIG-UTP, depending on the desired percentage of modification (for example, to obtain a 15% biotin and 10% DIG riboprobe: 75 μM biotin-UTP; 50 μM DIG-UTP, 375 μM UTP); 1 U/ml of ribonuclease inhibitor; 40 U of T7 or T3 RNA polymerase; 1 μg of plasmid DNA linearized after digestion with a suitable restriction endonuclease (EcoR1 or Hind III). The mixture is incubated for 2 hours at 37° C. When transcription is complete, the plasmid DNA is digested by adding 1 μl (1 unit) of DNase RQ1 (Promega). The reaction is incubated for 15 minutes at 37° C. The unincorporated ribonucleotides are removed by passage through a Biospin 30 exclusion chromatography column (Biorad).

Provenance & Evidence