Isolation of high molecular weight DNA for reliable genotyping of transgenic mice.
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 degrees 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.
Malumbres
M
M
New York University Medical Center, NY, USA.
Mangues
R
R
Ferrer
N
N
Lu
S
S
Pellicer
A
A
D000818:Animals; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D019286:Endopeptidase K / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018628:Gene Dosage; D005838:Genotype; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D019384:Nucleic Acid Synthesis Inhibitors; D016133:Polymerase Chain Reaction / Q000379:methods*; D013623:Tail; D019914:Taq Polymerase; D013698:Templates, Genetic; D014034:Toes
D017931:DNA Primers; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes; D019286:Endopeptidase K