Appendix 1. Protocol used for PCR amplification and DNA sequencing
Twenty-five overlapping fragments of the mitochondrial genome were amplified by
PCR using primers published in Hassanin et al. (2009), Hassanin et al. (2012) and Hassanin
(submitted a,b): U400M1/12SL200; U400M1/12SL600; 12SU1230/12SL2226M1LA;
12SU829/16SL518; 16SU365/16SL1056; 16SU946/N1L64; Uleu/LMet2;
N1U840/N2L492; N2U354M2/AsnL; TrpU/C1L705; UTyr/C1L1017;
C1U897M1/C2L15M1; SerU/A8L1; C2U603M4/C3L168; A6U654M1/GlyLM1;
C3U780M3 / N4L366M2; UArg/N4L918M1; N4U681/Leu2LM1; Ser2U/N5L652;
N5U501/N5L1214; N5U1146M5/N5L154M5; N6RU102/CBL402; GluMA/ProMA;
CBU162/L482; and UThr13/L482. Amplifications were done in 20 μl using 3 μl of Buffer
10X with MgCl2, 2 μl of dNTP (6.6 mM), 0.12 μl of Taq DNA polymerase (2.5 U, Qiagen,
Hilden, Germany) and 0.75 μl of the two primers at 10 μM. The standard PCR conditions
were as follows: 4 min at 94°C; 5 cycles of denaturation / annealing / extension with 45s at
94°C, 1 min at 60°C and 1 min at 72°C, followed by 30 cycles of 30s at 94°C, 45s at 55°C,
and 1 min at 72°C, followed by 10 min at 72°C. Both strands of PCR products were
sequenced using Sanger sequencing on an ABI 3730 automatic sequencer at the Centre
National de Séquençage (Genoscope) in Evry (France). The sequences were edited and
assembled using Sequencher 5.1 (Gene Codes, Corp., Ann Arbor, MI, USA).
References
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Hassanin A. (submitted). The complete mitochondrial genome of the Servaline Genet,
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