Cloning, production and characterization of wild type and mutant forms of the R·EcoK endonuclases

• Published in: Nucleic acids research 1993-04, Vol.21 (7), p.1686-1686
• Main Authors: Weiserova, M., Janscak, P., Benada, O., Hubácek, J., Zinkevich, V.E., Glover, S.W., Firman, K.
• Format: Article
• Language: English
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/21.7.1686

The publishers wish to apologize for a disk translation error which resulted in ‘μ’ being incorrectly replaced by ‘m’ in the Materials and Methods section of this paper. Please note that in Media and microbial procedures, ampicillin was used at a concentration of 50 μg/ml. In Protein production and analysis, the endonuclease activity was assayed in a volume of 100 μl that contained 8 μg of DNA, 0.8 μg of EcoK enzyme, 0.1 mM SAM and 50 μg/ml BSA. Immediately before, and at appropriate intervals after the addition of ATP, 5 μl aliquots were mixed with 1.5 μl of stop solution. The gels were photographed following staining with ethidium bromide (0.5 μg/ml). The methylase activity at 30°C and 42°C was measured in the same buffer used for the endonuclease reactions. The 370 μl of reaction mixture contained 2.4 μg of enzyme, 3.7 μg heteroduplex DNA, 1 μM [methyl3H]S-adenosyl methionine (85 Ci/mmol). ATP was added to a final concentration of 5 μM. At appropriate intervals, 50 μl aliquots were mixed with 5 μl 0.5M EDTA pH 8.0 and the samples were extracted with phenol-chloroform.