Effect of lambda packaging extract mcr restriction activity on DNA cloning

• Published in: Nucleic acids research Vol. 17; no. 13; p. 5409
• Main Authors: KRETZ, P. L, REID, C. H, SHORT, J. M
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 11.07.1989
• Subjects: Animals; Bacteriophage lambda - genetics; Biological and medical sciences; Biotechnology; Cloning, Molecular - methods; Cosmids; Cricetinae; DNA - genetics; DNA Restriction Enzymes; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; genes; Genetic engineering; Genetic technics; Genetic Vectors; Humans; Hybrid Cells; Methods. Procedures. Technologies; Methylation; restriction-modification; DNA; Escherichia coli; Cosmid; Restriction endonuclease; Packaging; Bacteria; Genetic engineering; Molecular cloning; Methylation; Escherichieae; Enterobacteriaceae
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/17.13.5409

The negative effect of bacterial mcrA and mcrB restriction activity on the cloning of methylated DNA has recently been demonstrated. In order to determine the effect of these restriction systems on lambda and cosmid packaging, a mcrA-, B-, hsd-, mrr- packaging extract strain was constructed by P1 transduction. The extract prepared from this strain, Gigapack II, was tested against the restriction positive (mcrA+,B-) extract, Gigapack I, by comparing efficiencies in constructing a cosmid library. The results indicate that these restriction enzymes are active in lambda packaging extracts and can affect cloning efficiencies. The elimination of mcrA,B restriction activity from Gigapack II allowed a 2-3 fold increase in the number of cosmid clones obtained.