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.
Biswas
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Department of Pathology and Biochemistry, University of Kentucky, Lexington 40536, USA.
D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D012097:Repressor Proteins