Polymerase chain reaction amplification of lacZ to engineer a gene sequence that can be used to modify the size of pUC19

Abstract

In an attempt to increase the size of pUC19, the lacZ gene (3kb) was targeted for amplification from Escherichia coli C29 genomic DNA by PCR. Gel electrophoresis showed that a 3kb band was absent, and major products about 700bp and 1500bp were present. To determine what had been amplified, PCR products were cloned into a vector prior to sequencing. None of the clones contained the 1500bp product. Sequence analysis showed that the 700bp fragment is the hisG gene. Possibly due to the large size of the template and poor primer design, the PCR primers annealed to a different location and the desired construct was not obtained.