NcoI R6513

Protocol tips

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	PCR products were digested with NcoI (Promega) overnight at 37°C. PCR products and restriction enzyme (RE) digests were visualized on ethidium bromide-stained 10% polyacrylamide gels.

Protocol tips
PCR products were digested with NcoI (Promega) overnight at 37°C. PCR products and restriction enzyme (RE) digests were visualized on ethidium bromide-stained 10% polyacrylamide gels.

Publication protocol

Genotyping the TNF-α −308 promoter region polymorphism was performed using a technique that introduces a NcoI restriction site into PCR products at the site where the polymorphism occurs.9 Primers for this reaction were: forward 5′-AGG CAA TAG GTT TTG AGG GCC AT-3′ and reverse 5′-TCC TCC CTG CTC CGATTC CG-3′. 25–50 ng of total DNA was amplified by PCR in a 25 μl reaction mixture containing 1.5 mM MgCl2, 50 mM KCl, 200 μM dNTPs, 500 nM primers and 1.25 U Taq polymerase. PCR reactions were run at 94°C for 5 min, followed by 40 cycles consisting of melting at 94°C for 3 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min. PCR amplifications included a final extension step at 72°C for 10 min to insure full-length transcripts. PCR products were digested with NcoI (Promega) overnight at 37°C. PCR products and restriction enzyme (RE) digests were visualized on ethidium bromide-stained 10% polyacrylamide gels. Patients homozygous for the G allele have an NcoI restriction site in the PCR products and therefore have two bands of 87 and 20 bp, patients homozygous for the A allele will have a single uncut 107 bp product and heterozygotes have all three bands.

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