URA3 Deletion Plan (update 6/1/2010) A. PCR reaction 1. PCR1: Template Yp101 Genomic DNA + URA3U5/URA3U3 2. PCR2: Template Yp101 Genomic DNA + URA3D5/URA3D3 3. PCR3: Template Yp101 Genomic DNA + HIS4-5/HIS4-3 Precipitate PCR products Digest PUC19 DNA with SmaI; Gel purify digested DNA B. Ligation: In each reaction, add 1ul digested PUC19 T-vector DNA, 1ul PCR product, 1mM ATP , 1ul T4 ligase, 25C for 2 hours. Transform fresh E coli competent cells (EC1, EC2, or EC3). C. Confirmation For PUC19-URA3UP: insert has two possible orientations PUC19-URA3-UP (Uncut: 4.2kb) Orientation 1: Hind III: 4.2 Kb Pvu II: 2.36, 1.34, 0.55 kb HindIII + Not I; 1.55, 2.66, 0.04 kb Orientation 2 Hind III: 4.2 kb Pvu II: 2.36, 1.23, 0.65 kb Hind III + Not I: 1.55, 2.66, 0.04 PUC19-URA3-Down (Uncut: 4.6kb)---Wang Ke Orientation 1 Pst I: 0.65, 3.95, 0.03 PvuII: 2.27, 2.36 kb EcoR I + Sbf I: 1.93, 2.66, EcoRI + HindIII: 1.98, 2.6 Orientation 2 Pst I: 1.31, 0.65, 2.67 PvuII: 2.27, 2.36 kb EcoR I + Sbf I: 1.93, 2.66, EcoRI + HindIII: 1.98, 2.6 PUC19-HIS4 (Uncut 5.76kb)---Chen Rong Orientation 1 Bgl II: 2.65, 3.11 kb BamHI: 5.09, 0.29, 0.38 Pst I: 2.67, 3.10 kb Pvu II: 2.36, 0.70, 2.69 kb Sbf I + Not I: 3.06, 2.67, 0.02kb Orientation 2 Bgl II: 2.65, 3.11 kb BamHI: 2.41, 2.97, 0.37 Pst I: 5.73, 0.03 kb Pvu II: 2.36, 2.80,0.59 kb Sbf I + Not I: 3.06, 2.67, 0.02kb D. Isolate three fragments: Digest PUC19-URA3UP with Hind III+ Not I---1.55 kb + 2.7 kb. Gel Isolate 1.55 kb fragment Digest PUC19-URA3Down with Sbf I + EcoR I---1.9kb + 2.7 kb. Gel Isolate 1.9kb fragment Digest PUC19-HIS4 with Not I+ Sbf I---3.0 kb + 2.7 kb, Gel Isolate 3.0 kb fragment. Digest PUC19 with HindIII+EcoR I---2.7 kb. Gel Isolate 2.7 KB fragment E. Ligation: PUC19 (2.7kb)+URA3UP(1.55kb)+HIS4 (3.0kb) + URA3Down (1.9kb) F. Confirmation of Clone: Uncut: 9.1kb Fist use BglII digestion : Bgl II: 2.6kb, 0.7kb, 0.2kb, 0.4kb, 5.1kb If confirmed, use following for the right cloneL PvuII: 2.3kb, 0.6kb, 1.6kb, 4.6kb KpnI: 3.3kb, 0.6kb, 5.2kb NcoI: 2.1 kb, 1.8kb, 1.9kb, 3.2kb 本文来源：https://www.wddqw.com/doc/c899e841021ca300a6c30c22590102020740f284.html