The ligations of various PCR products into AK3 were transformed and single colony PCR was carried out to check for correct ligation. The following PCR products were ligated:
EpsE 5' integration site (5'E, ~500bp)
EpsE 3' integration site (3'E, ~500bp)
Spectinomycin resistance gene (spec,800bp)
The pSB primers were used on the following cycle:
1 cycle - 95oC for 30 sec
30 cycle - 95oC for 30 sec, 60oC for 45 sec, 72oC for 1 min
The following colonies appear to have correct inserts: 5'E1, 3'E2, Spec1