Mutation and Cloning Efficiency

Mutation and cloning efficiency.

Increased cloning efficiency by temperature-cycle ligation.

Molecular cloning is a crucial, and often speed-limiting, step in many standard procedures of molecular biology. Ligation of cohesive DNA ends is normally carried out at 12–16 C to ensure a good balance between enzyme activity and stability of annealed DNA overhangs. Low temperatures generally reduce ligase activity, whereas too high temperatures may reduce cloning efficiencies by melting annea...

Improved efficiency M13 cloning using electroporation.

We compared the efficiency of transformation of recombinant M13 DNA obtained using the traditional calcium chloride method with that obtained by electroporatlon. M13 cloning experiments are often hampered by low yields I.e. insufficient numbers of recombinant plaques are generated. Low concentration of the target DNA, poor ligation due to target DNA impurity and small target fragment size are c...

From mutation mapping to phenotype cloning.

Current methods of detecting known mutations in genes are efficient and have in many instances been incorporated into the armamentarium of diagnostic medicine. Many genetic diseases, however, including autosomal dominant diseases with high mutation rates and genetically lethal X chromosome-linked diseases, result from heterogeneous and often private mutations in the respective genes. Methods of...

A high efficiency cloning and expression system for proteomic analysis.

The recent description of the complete genomes of the two most pathogenic species of Brucella opens the way for genome-based analysis of the antigenicity of their proteins. In the present report, we describe a bench-level high-efficiency cloning and expression system (HECES) that allow expression of large numbers of Brucella proteins based on genomic sequence information. Purified proteins are ...