Dnazol method

Genomic DNA was extracted from single larvae and adults of Cx. pipiens mosquitoes using the CethylTrymethyl Ammonium Bromide (CTAB) method (Navajas et al. 1999 ) and the DNAzol method (according to the manufacturer's instructions -Invitrogen) respectively. MANUSCRIPT An allele specific PCR was used to identify individuals carrying the I1043L mutation (Grigoraki et al. 2017) . The primers External F (5'-GCAGTCCTTCGGCGATCTT-3'),External_R(5'-GAACAGTCCGGCGATGGATA-3'),CTC F (5'-GGCTTGATCTACCTGCTGTCTC-3') and ATC R (5'-AACAGCAAGTACATAGACGGGAT-3') were combined in one reaction to amplify a control band (External_F/External 352 bp band), the susceptible allele(ExternalF/ATCR135bpband)andtheresistantallele(CTCF/ExternalR60bp band) respectively if present. The PCR conditions were initial denaturation for 5 min at 95°C followed by 28 cycles of 30 sec at 95°C, 30 sec at 68°C, 1 min at 72°C and a final extension of 5 min at 72°C. PCR products were analyzed by electrophoresis on a 2% agarose gel. Samples of known genotype were included in the analysis as positive controls and reactions without DNA were included as negative controls.