5'-Atp → Anthranilic Acid

Participants

Reaction Conditions

Reaction Steps

10 units of restriction enzyme SalI (product of Takara Shuzo Co.) was added to 200 μl of the SalI reaction solution containing 3 μg of pCE53 plasmid DNA prepared as above and 9 μg of the chromosomal DNA. The mixture was allowed to react to 37° C. for 60 minutes and heated at 65° C. for 10 minutes to stop the reaction. Then, 40 μl of the T4 ligase buffer (pH 7.6) consisting of 660 mM Tris, 66 mM MgCl2 and 100 mM dithiothreitol, 40 μl of 5 mM ATP, 0.4 μl of T4 ligase (product of Takara Shuzo Co., 1 unit/μl) and 120 μl of H2O were added to the digest. The mixture was allowed to react at 12° C. for 16 hours.

Provenance & Evidence