Sigmaspin sequencing reaction clean up columns

Additionally, part of the sodium dehydrogenase subunit 4 (nad4) gene of the mitochondrial DNA of the sampled specimens was sequenced using a modification of the protocol of Fonseca et al. [32 (link)]. The primers used, ND4F (5′-CGTAGGAGGAGCAGCTATATT-3′) and ND4R1X (5′-TGATTGCCTAAGGCTCATGT-3′) [33 (link)], amplify a 424 bp fragment between positions 8398 and 8821 in the Anopheles gambiae genomic sequence (GenBank accession no. L20934). DNA amplification was preceded by a 10 min denaturation step at 96°C and consisted of 35 cycles of 40 s at 94°C, 40 s at 56°C and 60 s at 72°C. A final extension step of 7 min at 72°C was added. PCR products were checked by electrophoresis on a 1.5% agarose gel run for one hour and visualised by ethidium-bromide staining. DNA bands were excised and recovered with the QIAamp Gel Extraction Kit (Qiagen). Afterwards, they were cycle-sequenced in both directions with the BigDye Terminator v1.1 Cycle Sequencing Kit (Life Technologies). PCR products were cleaned with SigmaSpin Sequencing Reaction Clean-Up Columns (Sigma-Aldrich) before being run on a 3130xl Genetic Analyzer. FASTA files of the obtained sequences were aligned with MultAlin [34 (link)] to detect nucleotide polymorphisms.

We sequenced a 424-bp fragment in the sodium dehydrogenase subunit 4 (nad4) region of the mosquito mitochondrial DNA (between positions 8398 and 8821 in the Anopheles gambiae complete mitochondrial genome sequence, GenBank accession #L20934, [8] (link)) that has shown to be variable and informative for population level analyses [8] (link). We used primers ND4F 5′-CGTAGGAGGAGCAGCTATATT-3′ and ND4R1X 5′-TGATTGCCTAAGGCTCATGT-3′[40] . Amplifications were performed in a Bio-Rad C1000 thermal cycler (Bio-Rad Laboratories). Amplifications were preceded by a five minute denaturation at 96°C and consisted of 35 cycles of 40 s at 94°C, 40 s at 56°C and 40 s at 72°C, followed by a final extension step of five minutes at 72°C. PCR products were gel-electrophoresed, excised from the gels and recovered with a QIAamp Gel Extraction Kit (Qiagen). They were then cycle-sequenced in both directions with a BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems/Life Technologies) using one of the amplification primers, then purified with SigmaSpin Sequencing Reaction Clean-Up Columns (Sigma-Aldrich) and run on a 3130 Genetic Analyzer (Applied Biosystems/Life Technologies).

Detection of specific gene transcripts were performed as previously described in Brown et al., 2005 (link). Probes against Nkx3-2, Rab28, Bod1l and Gli3 were amplified from cDNA from wildtype C57BL/6NJ mouse embryos (Supplementary file 5). Amplified fragments were cloned into pJET1.2/blunt plasmid backbones in both sense and anti-sense orientations using the CloneJET PCR Kit (Thermo Fisher Scientific, Schwerte, Germany) and confirmed by Sanger sequencing using the included forward and reverse primers. Probe plasmids have also been deposited with Addgene. In vitro transcription from the T7 promoter was performed using the MAXIscript T7 in vitro Transcription Kit (Thermo Fisher Scientific) supplemented with Digoxigenin-11-UTP (Sigma-Aldrich) (MPI Tübingen), or with T7 RNA polymerase (Promega) in the presence of DIG RNA labeling mix (Roche) (University of Calgary). Following TURBO DNase (Thermo Fisher Scientific) digestion, probes were cleaned using SigmaSpin Sequencing Reaction Clean-Up columns (Sigma-Aldrich) (MPI Tübingen), or using Illustra MicroSpin G-50 columns (GE Healthcare) (University of Calgary). During testing of probe designs, sense controls were used in parallel reactions to establish background non-specific binding.