Open AccessIsolation of High Molecular Weight DNA for Reliable Genotyping of Transgenic Mice
Author(s) -
Marcos Malumbres,
Ramón Mangues,
Neus Ferrer,
Suying Lü,
Ángel Antonio Blasco Pellicer
Publication year - 1997
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/97226st03
Subject(s) - dna extraction , microbiology and biotechnology , dna , biology , proteinase k , polymerase chain reaction , genotyping , transgene , primer dimer , centrifugation , multiple displacement amplification , isolation (microbiology) , multiplex polymerase chain reaction , biochemistry , gene , genotype
A fast and reliable method for the PCR characterization of DNA from mouse toes is described. The toes biopsied to tag the mice are incubated for 2 h in proteinase K and heated for 15 min at 95°C. This DNA solution is directly used as a template for PCR amplification. The same procedure can be used for PCR analysis of DNA from other tissues in adult mice, mouse embryos and cultured cells. Because of minimal tissue manipulation, high-quality and high-molecular-weight DNA (fragments larger than 100–200 kb) is isolated. This procedure is performed in a single tube and requires no organic solvent extraction or centrifugation, allowing the isolation of high-molecular-weight DNA suitable for PCR amplification in a fast and reproducible way. Only the tissue excised during mice tagging is used and a large number of animals can be quickly and simultaneously analyzed as required to maintain a transgenic mice colony. In addition, this rapid and efficient procedure represents an alternative to other methods in which, in our experience, inhibition of the PCR amplification occurs when DNA from tail tissues is used.