DNA Extraction and Southern Blot Analysis of Insects

DNA was extracted from single female and male pupae using CTAB DNA extraction as described elsewhere [52 (link)]. Concentrations were measured on a Qubit 3.0 Fluorometer using the dsDNA BR Assay Kit (Invitrogen, Carlsbad, CA), and approximately 4 μg DNA per sample was used for DNA digestion reactions. DNA was double digested using NdeI × NotI, DraI × NheI (all Fermentas, Vilnius, Lithuania), or AgeI × BspHI (New England Biolabs, Ipswich, MA) enzymes (see S1 Methods for details). All digested DNA was separated by electrophoresis on a 1% TBE agarose gel and subsequently transferred by capillary transfer to an Amersham Hybond-N+ nylon membrane (GE Healthcare, Milwaukee, WI).
A probe specific to EkMasc with high homology to EkMascB (96%) was made by PCR-labeling (see S1 Methods for details). The probe was labeled with digoxigenin-11-dUTPs (Roche Diagnostics, Mannheim, Germany) using primers Masc_Sb_F and Masc_Sb_R. For Southern hybridization, 100 ng of the probe was used. The Southern blot assay was performed as described previously [55 (link)] with some modifications [56 (link)].