A general method to optimize the amount of enzyme in restriction and DNA modification reactions using the beta galactosidase blue-white plaques assay.
We propose a simple and economical method for assaying the activity of restriction and other modifying enzymes. The method involves assaying the use of the blue and white colored phenotypes of bacterial colonies obtained by digesting the polylinker sequence of M13 bacteriophage vectors followed by transformation in appropriate strains on X-gal/IPTG plates. In conjunction with restriction enzymes and DNA ligases, the method can evaluate polymerase activity and can be applied to test 3'...5' exonuclease activities such as that of T4 DNA polymerase, without having to use expensive radioisotopes. We describe its application in the assessment of restriction enzymes, DNA ligase and DNA polymerase activities.
Cab-Barrera
E L
EL
U.L.I.E.G.-Departamento de Bioquímica Facultad de Medicina, Monterrey, N.L. Mexico.
Barrera-Saldaña
H A
HA
D001435:Bacteriophages / Q000235:genetics; D001709:Biotechnology; D011088:DNA Ligases / Q000032:analysis; D015280:DNA Restriction-Modification Enzymes / Q000032:analysis*; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005696:Galactosidases / Q000235:genetics*; D005822:Genetic Vectors; D010641:Phenotype; D010858:Pigmentation; D014170:Transformation, Genetic; D010948:Viral Plaque Assay / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics*
D015280:DNA Restriction-Modification Enzymes; D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase; D005696:Galactosidases; D001616:beta-Galactosidase; D011088:DNA Ligases