Spermidine → Biotin
Reaction equation
CCOC(OC([O:9][CH2:10][CH3:11])=O)=O.C(O)[C:13]([NH2:18])([CH2:16]O)[CH2:14]O.Cl.[Mg+2].[Cl-].[Cl-].NCCCCN[CH2:30][CH2:31][CH2:32]N.[Na+].[Cl-].[CH2:36]([SH:43])[C@@H](O)[C@H](O)CS.P(OC[C@H]1[O:62][C@@H:61]([N:63]2C3N=CN=C(N)C=3N=C2)[C@H](O)[C@@H]1O)(OP(OP(O)(O)=O)(O)=O)(=O)O.P(OC[C@H]1O[C@@H](N2C3N=C(N)NC(=O)C=3N=C2)[C@H](O)[C@@H]1O)(OP(OP(O)(O)=O)(O)=O)(=O)[OH:76].C1C(=O)NC(=O)N([C@@H]2O[C@H](COP(OP(OP(O)(O)=O)(O)=O)(O)=O)[C@@H](O)[C@H]2O)C=1.OC(CCCC[C@H]1[C@@H]2[C@@H](NC(N2)=O)CS1)=O.C1C(=O)NC(=O)N([C@@H]2O[C@H](COP(OP(OP(O)(O)=O)(O)=O)(O)=O)[C@@H](O)[C@H]2O)C=1>>[OH:76][C:10]([CH2:11][CH2:32][CH2:31][CH2:30][C@H:16]1[C@@H:13]2[C@@H:14]([NH:63][C:61]([NH:18]2)=[O:62])[CH2:36][S:43]1)=[O:9]Participants
Reactant 10
Product 1
Procedure
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).
Sources & evidence
- ORD record
- ord-2f5423ea81744f7b88e2042fff66c043
- Source dataset
- uspto-grants-2001_05
- Patent number
- US06228580B1
Reaction notes
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