Genotype identification of Math1/LacZ knockout mice based on real-time PCR with SYBR Green I dye

Valery Krizhanovsky, Esther Golenser, Nissim Ben-Arie*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

Knockout mice are widely used in all fields of biomedical research. Determining the genotype of every newborn mouse is a tedious task, usually performed by Southern blot hybridization or Polymerase Chain Reaction (PCR). We describe here a quick and simple genotype identification assay based on real-time PCR and SYBR® Green I dye, without using fluorescent primers. The discrimination between the wild type and targeted alleles is based on a PCR design that leads to a different melting temperature for each product. The identification of the genotype is obvious immediately after amplification, and no post-PCR manipulations are needed, reducing cost and time. Therefore, while the real-time PCR amplification increases the sensitivity, the fact that the reactions tubes are never opened after amplification, reduces the risk of contamination and eliminates errors, which are common during the repeated handling of dozens of samples from the same mouse line. The protocol we provide was tested on Math1 knockout mice, but is general, and may be utilized for any knockout line and real-time thermocycler, without any further modification, accessories or special reagents.

Original languageAmerican English
Pages (from-to)187-192
Number of pages6
JournalJournal of Neuroscience Methods
Volume136
Issue number2
DOIs
StatePublished - 30 Jul 2004

Bibliographical note

Funding Information:
We thank Drs. Uri Gat and Allan Bar-Sinai for carefully reading the manuscript and Theodora Bar-El for the valuable assistance. This work was supported by grants from the US-Israel Binational Science Foundation (2001-013), the Israel Science Foundation (587/02), the European Community (QLG3-CT-2000-00072), and the Roland Center for Neurodegenerative Diseases.

Keywords

  • Genotype
  • Knockout mice
  • LacZ
  • Math1
  • Melting curve
  • Real-time PCR
  • SYBR Green I

Fingerprint

Dive into the research topics of 'Genotype identification of Math1/LacZ knockout mice based on real-time PCR with SYBR Green I dye'. Together they form a unique fingerprint.

Cite this