Cloning of PCR synthesized 191 bp DNA overlapping lac promoter for plasmid construction.

E coli genomic DNA was amplified by polymerase chain reaction (PCR) using two 5' and 3' oligonucleotide primers (27-mer). Amplified DNA was 191 bp. The region of amplified DNA on lac operon in E coli was 14 bp upstream from the transcription initiation site and 177 bp downstream. Amplified DNA was cloned into a phagemid vector for construction of plasmid, suitable for use as template for making strand-specific probe to detect initiated lac transcript by RNase protection assay, labelling for Southern and Northern hybridization. Another use of this probe made from this construct is to detect strand-specific DNA repair. The construct was verified by DNA sequencing.