Abi 3730 dna analyser

Total RNA from 6x10⁵cells/mL from hybridomas producing anti-LT and anti-ST monoclonal antibodies, previously described [17 (link), 18 (link)], were extracted by RNeasy Mini Kit (QIAGEN, Germany) following manufacturer recommendations, and 1 μg was reverse transcribed using random hexamer primers by using first-strand cDNA Synthesis kit (GE Healthcare, USA). DNA fragments corresponding to heavy and light chains variable domains were obtained by PCR amplification using Mouse ScFv Module of Recombinant Phage Antibody System kit (GE Healthcare, USA), cloned into pGEM-T Easy Vector (Promega, USA) and sequenced using M13 and SP6 sequencing primers, on ABI 3730 DNA Analyser (Life Technologies, USA). The obtained sequences were analyzed using BLAST and both scFv-LT and scFv-ST were designed by joining coding sequences of heavy-chain variable domain (VH) and light-chain variable domain (VL) with an intermediary flexible linker (Gly₄Ser)₃, in the VH-linker-VL orientation. Based on designed sequences, optimized genes were synthesized for expression in E. coli (GeneArt, Germany).

We determined individual bee genotypes at five microsatellite loci according to the protocol described by Evans et al. (2013 ) using the primers detailed in Supplementary Table S7. These microsatellite loci have been used in other honeybee population genetic studies (Muñoz et al., 2009 , 2012 ). We ran the microsatellites as two separate multiplexes, with primers A113, AP043, and AP055 in multiplex 1 and primers A007 and B124 in multiplex 2, using the Qiagen Type‐It master mix kit (Qiagen, Valencia, CA) in a final reaction volume of 10 μl. PCR amplification involved denaturation at 94°C for 5 min, followed by 30 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 30 s, with a final elongation step at 72°C for 30 min. The size of the microsatellites was determined by capillary electrophoresis on an ABI 3730 DNA Analyser (Applied Biosystems, CA, USA) at the University of Manchester Sequencing Facility, using the GeneScan™ LIZ500 (Thermo Fisher Scientific, USA) size standard. The output peaks were scored using Genemapper v5.0 software (Applied Biosystems) and sorted into allele bins using the MsatAllele package v1.05 (Alberto, 2009 (link)) in R (R Core Team, 2017 ). A subset of samples were repeated to ensure consistency of allele calling. The function hw.test in the package pegas (Paradis, 2010 (link)) was used to test whether the alleles were in Hardy–Weinberg equilibrium.