FastDigest XagI

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	A volume of 10 μl of PCR products was digested for 1 h at 37°C with XagI (FastDigest, Fermentas) for IL-17A G197A and NlaIII (FastDigest, Fermentas) for IL-17F A7488G.	The digested PCR products were electrophoresed on 5% (w/v) agarose gel prestained with ethidium bromide at 70 V for 50 min and visualized using the FluorChem 5500 imaging system (Alpha Innotech Corp., San Leandro, CA, USA).

Protocol tips
A volume of 10 μl of PCR products was digested for 1 h at 37°C with XagI (FastDigest, Fermentas) for IL-17A G197A and NlaIII (FastDigest, Fermentas) for IL-17F A7488G.
Downstream tips
The digested PCR products were electrophoresed on 5% (w/v) agarose gel prestained with ethidium bromide at 70 V for 50 min and visualized using the FluorChem 5500 imaging system (Alpha Innotech Corp., San Leandro, CA, USA).

Publication protocol

"Genotyping analysis of IL-17A G197A and IL-17F A7488G polymorphisms was conducted using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method. Primer sequences for IL-17F A7488G were adopted from a previous study [36] and as follows: (forward) 5'-ACCAAGGCTGCTCTGTTTCT-3' and (reverse) 5'-GGTAAGGAGTGGCATTTCTA-3'. The primer sequences for IL-17A G197A (forward: 5'-GGAACATGAATTTCTGCCCTTCCCA-3'; reverse: 5'-TAGGGCTTTTCTCCTTCTGTGGTCA-3') were designed using the NCBI Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Genomic PCR was performed in a 30-μl reaction volume containing 30 ng genomic DNA, 2.0 mM MgCl2, 0.2 mM of each deoxynucleotide triphosphate, 0.3 μM of each primer (forward and reverse), 1 U Taq DNA polymerase, and 1X Taq buffer with KCl (Fermentas; Thermo Fisher Scientific, Glen Burnie, MD, USA), using the Mastercycler gradient thermal cycler (Eppendorf, Hamburg, Germany). The thermal cycling conditions comprised an initial denaturing step at 94°C for 5 min and subsequent 30 cycles of denaturation at 94°C for 30 s, annealing at 54°C for IL-17A and 60°C for IL-17F for 30 s, and extension at 72°C for 30 s. A final extension at 72°C for 10 min was performed.

A volume of 10 μl of PCR products was digested for 1 h at 37°C with XagI (FastDigest, Fermentas) for IL-17A G197A and NlaIII (FastDigest, Fermentas) for IL-17F A7488G. The digested PCR products were electrophoresed on 5% (w/v) agarose gel prestained with ethidium bromide at 70 V for 50 min and visualized using the FluorChem 5500 imaging system (Alpha Innotech Corp., San Leandro, CA, USA)."

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