Improvement in the visual discrimination of recombinant clones by size reduction of non-recombinant colonies

• Published in: Journal of microbiological methods Vol. 95; no. 2; pp. 302 - 303
• Main Authors: Sektas, Marian, Furmanek-Blaszk, Beata
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2013
• Subjects: agarose; clones; Cloning, Molecular - methods; DNA; DNA methyltransferase; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; DNA-Directed DNA Polymerase - genetics; Escherichia coli - genetics; Escherichia coli - isolation & purification; gels; genes; Genetic Vectors; methyltransferases; Mutagenesis, Insertional; Positive/negative screening; Recombinant technology; toxicity; Positive/negative screening; DNA methyltransferase; Recombinant technology
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2013.09.003

A flexible approach circumventing cloning problems related to incomplete vector double digest is described. DNA methyltransferase gene insertion into MCS of commonly used expression vectors facilitates identification of both: i) the correct linear fragment in agarose gels due to the dilator effect, and ii) recombinant colonies by size and opacity differences resulting from methyltransferase toxicity. •A novel versatile method helps to improve cloning efficiency and recombinants screening is described.•A modification of multiple cloning site of vector by toxic gene is proposed.•Replacement of the toxic gene on target gene facilitates identification of true recombinants.•Screening of recombinants colonies relies on size and opacity differences.•This approach may be adapted and customized to any other expression vector.