Appendix H. Methods for molecular sexing of chicks and embryos.
To sex tit nestlings, we used a DNA test following the protocol of Griffiths et al. (1998). DNA was extracted from nestling’s blood using commercial kits (Wizard Genomic DNA Isolation Kit, Promega, Switzerland; DNeasy Tissue Kit, Qiagen, Basel, Switzerland) following the manufacturers' protocols. For DNA extraction from dead embryo tissue, we used DNeasy Tissue Kit (Qiagen, Basel, Switzerland), following the protocol for rodent tail.
PCR amplification was carried out in a total volume of 10 µL. The final reaction conditions were as follows: 0.25 U HotStarTaq DNA polymerase (Qiagen, Basel, Switzerland), 1 µL Taq buffer, 2.5 mM MgCl2, 0.2 mM of each dNTP (Amersham Pharmacia Biotech Inc.) and 1 µM each of primers P2 and P8. 1 µL of genomic DNA was used as template. PCR was performed in a GeneAmp 2400 or GeneAmp 9700 Thermocycler (Applied Biosystems, Rotkreuz, Switzerland) with the following temperature profile: initial denaturation at 95°C for 15 min; 40 cycles of 94°C for 30 sec, 52°C for 15 sec, and 72°C for 75 sec. The program was completed by an additional extension step at 72°C for 7 min.
PCR products were separated by electrophoresis at 8 V/cm on ethidium bromide stained 2% agarose gels and visualized by UV transillumination. The nestlings were sexed according to the presence of one (males) or two bands (females) (Tschirren et al. 2003).
LITERATURE CITED
Griffiths, R., M. C. Double, K. Orr, and R. J. G. Dawson. 1998. A DNA test to sex most birds. Molecular Ecology 7:1071–1076.
Tschirren, B., P. S. Fitze, and H. Richner. 2003. Sexual dimorphism in susceptibility to parasites and cell-mediated immunity in great tit nestlings. Journal of Animal Ecology 72:839–845.