MHC-IIB amplicon sequencing of black-legged kittiwakes: blood
DNA was extracted from blood samples using the blood and tissue DNA extraction kit (QIAGEN). A 258-pb fragment of exon 2 of MHC classIIB was amplified using specific primers (forward: 5- GCAYGAGCAGGGTATTTCCA, and reverse: 5-GTTCTGCCACACACTCACC). The 5 end of both forward and reverse primers included a combination of two different 8-bp tags. The PCR amplification was performed in 20 l mixtures 1  AmpliTaq Gold 360 Master Mix (Applied Biosystems) and 3.2 g containing 2 l of extracted DNA, 0.5 m of each primer, bovine serum albumin (Roche Diagnostics). The PCR programme consisted of 10 min initial denaturation at 95C, followed by 30 cycles of 30 s denaturation at 95C, 30 s annealing at 57C and 90 s extension at 72C. A final elongation step was run at 72C for 7 min. Amplicons were then pooled, purified using the MinElute PCR Purification protocol (Qiagen), and sent for sequencing to Fasteris (Switzerland) in March 2024. They were sequenced using the 2x300-bp protocol.