FastDigest XapI

Protocol tips

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	The amplified product was digested with FastDigest XapI restriction endonuclease according to manufacturer's recommendations (Thermo Fisher Scientific, Lithuania). Allele-specific RFLP products were separated by electrophoresis on a 1–2% agarose gel and 0.5xTris/Borate/EDTA buffer.

Protocol tips
The amplified product was digested with FastDigest XapI restriction endonuclease according to manufacturer's recommendations (Thermo Fisher Scientific, Lithuania). Allele-specific RFLP products were separated by electrophoresis on a 1–2% agarose gel and 0.5xTris/Borate/EDTA buffer.

Publication protocol

"In order to exclude amplification of other highly homologous hGH/CSH genes, the genotyping protocol of GH2 rs2006123 SNP included long-range PCR to amplify specifically the entire GH2 gene region (5634 bp product [16]; forward primer: 5′-AGGTCTGGAAAGGAGGAGACAAAAGAG-3′, reverse primer: 5′-TTGAATTAGACTTGGGATTCTCCTGACA-3′), and allele-specific restriction fragment length polymorphism (RFLP) analysis (Supplemental Figs. S1–S2). In case the long-range PCR had resulted in insufficient product quantity for the RFLP detection, nested PCR (1855 bp product; forward primer: 5′-GCTTGTGGTCCGTGTCTGTTCC-3′, reverse primer: 5′-GGTGGCGATAGACGTTGCTGTCC-3′) was applied to further amplify the DNA fragment involving the targeted SNP. PCR conditions are described in Supplemental Text S1.

The amplified product was digested with FastDigest XapI restriction endonuclease according to manufacturer's recommendations (Thermo Fisher Scientific, Lithuania). Allele-specific RFLP products were separated by electrophoresis on a 1–2% agarose gel and 0.5xTris/Borate/EDTA buffer. The following fragments were detected in the RFLP analysis of (i) long-range PCR: 2658, 1626, 541, 484, 218, 107 bp (C-allele); 1626, 1614, 1044, 541, 484, 218, 107 bp (A-allele) (Supplemental Fig. S1A) or (ii) nested PCR: 1546 and 309 bp (C-allele); 1045, 501, and 309 bp (A-allele) (Supplemental Fig. S1B). In developmental phase of the RFLP-based genotyping assay, a subset of samples were sequenced for the quality control and to verify few ambiguous genotypes using previously published sequencing primers and conditions [16]."

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Manufacturer protocol

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