MlsI (MscI) (5 U/µL)

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	The PCR products were digested with fast digest MscI (Thermo Scientific) according to the manufacturer’s instructions and analyzed on a 3% agarose gel.

Protocol tips
The PCR products were digested with fast digest MscI (Thermo Scientific) according to the manufacturer’s instructions and analyzed on a 3% agarose gel.

Publication protocol

Genomic DNA was extracted from the samples of peripheral venous blood using salting out and method by V Iranpur M [15, 16]. The G → T polymorphism in the Sp1 binding site in the COLIA1 gene was examined by a polymerase chain reaction (PCR). A mismatched primer was used which introduces a restriction site for the enzyme Msc I in the polymorphic alleles with the T substitution that discriminates S and s allele, which corresponding the presence of G and T respectively. Thus, the absence of the restriction site on both alleles was defined as SS homozygote and the presence of the restriction site ss homozygote whereas the combination of as Ss heterozygote [6]. The forward primer, 5′-TAACTTCTGGACTATTTGCGGACTTTTTGG-3 and the reverse primer, 5′-GTCCAGCCCTCATCCTGGCC-3″ were used for PCR in a final volume of 50 µl containing 0.1–0.2 mg of genomic DNA and others Taq DNA polymerase (Ampli Taq 360 DNA Polymerase as per protocol of Applied Biosystem, USA) using standard conditions on an GENEI TC-3000 thermal cycler (Bangalore Genei, India) for 35 cycles at 94 °C (50 s), 61 °C (30 s) and 72 °C (60 s). Prior to the first cycle, initial denaturation was performed at 94 °C for 4 min and the last extension step with 5 min. The PCR products were digested with fast digest MscI (Thermo Scientific) according to the manufacturer’s instructions and analyzed on a 3% agarose gel. The PCR product containing s alleles are cleaved by MscI, resulting in a band of 246 bp compared with the un-cleaved S alleles band of 264 bp. Agarose gel electrophoresis of PCR amplified products and restriction MscI-digested fragment are shown in Fig. 1.

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