MspA1I NEB#R0577

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	A volume of 10 μl of each polymerase chain reaction (PCR) product was digested with 2 U of MspA1I (10 U/μl, NEB, Izasa, Barcelona, Spain), 2 μl 10 × buffer 1, 0.15 μl bovine serum albumin (100 × ) and 7.65 μl H2O for 5 h at 37°C.	Digests were then electrophoresed on a 3% metaphor agarose gel, which resulted in four fragments of 85, 67, 40 and 15 bp (allele T) or in five fragments of 73, 67, 40, 15 and 12 bp (allele C), respectively.

Protocol tips
A volume of 10 μl of each polymerase chain reaction (PCR) product was digested with 2 U of MspA1I (10 U/μl, NEB, Izasa, Barcelona, Spain), 2 μl 10 × buffer 1, 0.15 μl bovine serum albumin (100 × ) and 7.65 μl H2O for 5 h at 37°C.
Downstream tips
Digests were then electrophoresed on a 3% metaphor agarose gel, which resulted in four fragments of 85, 67, 40 and 15 bp (allele T) or in five fragments of 73, 67, 40, 15 and 12 bp (allele C), respectively.

Publication protocol

Genomic DNA was extracted from ethlyenediamine-tetraacetic acid-preserved whole blood using the QIAamp DNA Blood Mini extraction kit (Qiagen, Izasa, Barcelona, Spain). The region containing the polymorphic sites at positions +869 (T10 → C10, Leu → Pro) (refSNP ID: rs1982073) and +915 (G25 → C25, Arg → Pro) (refSNP ID: rs1800471) in the first exon of the TGFB1 gene was amplified according to previously described methods.21 A volume of 10 μl of each polymerase chain reaction (PCR) product was digested with 2 U of MspA1I (10 U/μl, NEB, Izasa, Barcelona, Spain), 2 μl 10 × buffer 1, 0.15 μl bovine serum albumin (100 × ) and 7.65 μl H2O for 5 h at 37°C. Digests were then electrophoresed on a 3% metaphor agarose gel, which resulted in four fragments of 85, 67, 40 and 15 bp (allele T) or in five fragments of 73, 67, 40, 15 and 12 bp (allele C), respectively. For typing the codon 25 polymorphism at position +915 (G → C), 10 μl of the PCR products were digested with 2 U of Sau96I (Roche) for 5 h at 37°C. Allele C was defined by the presence of two fragments of 113 and 94 bp and allele G by the presence of an intact fragment of 207 bp.

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