Sequencing of the full coding sequence of cxcr3.1, cxcr3.2 and cxcr3.3 was obtained by amplification with primers described in supplementary material Table S3 . For cxcr3.2 and cxcr3.3, both genomic and cDNA templates extracted from pools of 15–20 embryos were used. Amplification was performed with Phusion high-fidelity DNA polymerase (Thermo-Scientific, Pittsburgh, PA, USA). DNA amplicons were then gel-extracted on 1.5% agarose and column-purified with PureLink quick gel extraction and PCR purification kit (Invitrogen, Life Technologies, Carlsbad, CA, USA). Sequencing with M13Fw, M13Rv universal primers (incorporated in the amplification primers) or with custom-made primers was outsourced to Baseclear (Leiden, The Netherlands). For cxcr3.1, sequencing results derive exclusively from genomic DNA amplifications. Amplification of cDNA templates for cxcr3.2 resulted in a band of identical size in mutant, wt and AB/TL, thereby excluding altered exon/intron arrangements attributable to the ENU-mutagenesis per se or to the cxcr3.2hu6044 allele.
Purelink quick gel extraction and pcr purification kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PureLink Quick Gel Extraction and PCR Purification Kit is a laboratory product designed to purify DNA fragments from agarose gels and to clean up PCR reactions. The kit utilizes a silica-based membrane technology to selectively bind DNA while removing contaminants. It provides a simple and efficient method for DNA extraction and purification.
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Lab products found in correlation
2 protocols using purelink quick gel extraction and pcr purification kit
1
Sequencing of Zebrafish CXCR3 Genes
2
16S rRNA Amplification and Sequencing
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Degenerate primers were used for the 16S rRNA PCR reaction, described by Weisburg et al. (1991) (link). Both the FD1 forward and RD1 reverse, and the FD1 forward and the RP1 reverse primer sets were used. Using each primer set on two different template concentrations (5-12 ng/μl), a total of four PCR reactions were performed. Amplified fragments were gel purified using the PureLink Quick Gel Extraction and PCR purification kit (Invitrogen), and sequenced by Sanger sequencing using the respective FD1, RD1 and RP1 sequencing primers (at the Iowa State University DNA core facility). The degenerate primers were designed to amplify most bacterial 16S rRNA and given the known impurities in the culture this could result in the amplification of 16S rRNA from different species, however all of the sequencing reactions gave the same result (based on CLUSTALW alignment of the sequenced fragments). Forward and reverse sequences were assembled into one 16S rRNA sequence of 1494 bp. This was used to BLAST against the assembled Tsp. jenense DSM 216 T genome, which identified the complete 16S rRNA sequence (located near the end of contig 73).
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