Sequence of PCR amplicon created using bombicar primers for partial small ribosomal subunit of Nosema bombi
Removed abdominal contents from Bombus occidentalis specimen. Homogenized with 100 uL sterile water. Extracted DNA by first using CTAB lysis buffer, then phenol and chloroform followed by ethanol. PCR was performed using bombicar primers (Plischuk et al. 2009). Cleaned up PCR product using Exonuclease 1 and shrimp alkaline Phosphotase (EXOSAP). Performed Big Dye sequencing reaction. Cleaned up reaction products by salting out and ethanol precipitation, then resuspended DNA in formamide, denatured and finally sequenced.