JL081406-Construction of J23067,J23068,J23069 for traG knockout

 PCR JL20/JL21 on RP4 gen.   (2268 bp, MfeI/BglII) Sub into pGLN           (BamHI/EcoRI) Product is pJL20(J23067)

IPCR JL22/JL23 on pJL20(J23067)   (3454 bp, EcoRI/PstI) Sub in pSB1A2-Bca9007       (1015 bp, EcoRI/PstI) Product is pJL22(J23068)

PCR JL18/JL19 on pJL22(J23068) for D/W  (bp, DpnI) Datsenko Wanner on pKD46/Rlambda, Product is J23069

JL20   Forward BglII to BamHI for traG-5' knockout cassette GAGTCAGATCTgattatgttggtgcgctg JL21   Reverse MfeI to EcoRI for traG-5' knockout cassette gctggCAATTGcattcgatttccagtacaggc JL22   Reverse EcoRI IPCR of traG gctgcGAATTCgatactggaaggaatgggc JL23   Forward PstI IPCR of traG cattgCTGCAGacggcagcattgaagaggcg