Resources for non-standard genotyping
Quantitative (real-time) PCR (qPCR)
Method that quantifies DNA sequences by normalizing to a reference gene, or to the amount of DNA in the reaction. QPCR is the easiest way to differentiate homozygous (Tg/Tg) from hemizygous (Tg/0) transgenic mice or to determine transgene copy number. For more information:
- Applied Biosystems - Tutorials and Troubleshooting
- Real-time polymerase chain reaction (Wikipedia)
- Taqman (Wikipedia)
Melting curve analysis
Method that uses the melting point of double-stranded PCR products to determine the size of the DNA fragments amplified for transgenes and knock-ins (eliminating the need for agarose gels). This requires a fluorescent dye (typically SYBR green) and special thermocycler (e.g. LightCycler® 480 from Roche). Melting curve analysis protocols provided on JAX® Mice datasheets can be used as a starting point for standard PCR, by eliminating the SYBR green and optimizing the protocol for use in a standard thermocycler. More information on melting curve analysis:
Pyrosequencing
Sequencing method that uses a chemiluminescent enzyme to detect specific nucleotide incorporation into DNA to genotype knockout and spontaneous mutants. For more information:
- Pyrosequencing (Wikipedia)
- Pyrosequencing (Youtube)
- Fakhrai-Rad et al. (2002). "Pyrosequencing: an accurate detection platform for single nucleotide polymorphisms". Hum Mutat. 19: 479. PMID 11968080
LacZ staining
Quick protocol for tail samples:
