BglI R6071

Protocol tips

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	PCR products were digested with BglI (Promega) for 2 h at 37°C. BglI digestion yields 131, 103 and 60 bp fragments in patients homozygous for the G allele, 163 and 131 bp fragments in patients homozygous for the C allele, and all four fragments in heterozygotes.

Protocol tips
PCR products were digested with BglI (Promega) for 2 h at 37°C. BglI digestion yields 131, 103 and 60 bp fragments in patients homozygous for the G allele, 163 and 131 bp fragments in patients homozygous for the C allele, and all four fragments in heterozygotes.

Publication protocol

Genotyping the TGF-β1 +915 polymorphism was accomplished by PCR amplification of genomic DNA using primers that span the polymorphic region followed by restriction analysis.37 Primers for this reaction were: forward 5′-TTC-CCT-CGA-GGC-CCT-CCT-A-3′ and reverse 5′-GCC-GCA-GCT-TGG-ACA-GGA-TC-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, 0.1% Tween 20 and 1.25 U Taq polymerase. Cycling parameters were: initial melting at 96°C for 10 min, followed by 35 cycles of melting at 96°C for 1 min, annealing at 62°C for 1 min and extension at 73°C for 1 min. PCR reactions included a final extension step at 73°C for 10 min. PCR products were digested with BglI (Promega) for 2 h at 37°C. BglI digestion yields 131, 103 and 60 bp fragments in patients homozygous for the G allele, 163 and 131 bp fragments in patients homozygous for the C allele, and all four fragments in heterozygotes.

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