MunI (MfeI) (10 U/µL)

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	The mutation in the fifth exon of the NBN gene (p.I171V) was assessed by restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) products, using MunI (MfeI) restriction enzyme (Fermentas, Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA).

Protocol tips
The mutation in the fifth exon of the NBN gene (p.I171V) was assessed by restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) products, using MunI (MfeI) restriction enzyme (Fermentas, Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA).

Publication protocol

DNA was extracted from blood using the columns provided in the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's instructions, and from Guthrie cards, as described previously (13,15). The mutation in the fifth exon of the NBN gene (p.I171V) was assessed by restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) products, using MunI (MfeI) restriction enzyme (Fermentas, Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA). PCR reactions were performed in a total volume of 25 µl containing 2.5 µl of 10X PCR buffer with 15 mM MgCl2 (Sigma-Aldrich, St. Louis, MO, USA), 6 pM of each primer, 2 mM of each deoxynucleotide triphospahate (dNTP; Sigma-Aldrich, Steinheim, Germany), 1.5 units of Taq DNA polymerase (Sigma-Aldrich, St. Louis, MO, USA) and 1.5 µl of DNA template. Amplification conditions involved initial denaturation for 5 min at 95°C, followed by 35 cycles of 25 sec at 95°C, 35 sec at 54°C and 55 sec at 72°C, with a 10-min final extension at 72°C. Next, 10 µl of the PCR products were digested for 3 h at 37°C, and analyzed by electrophoresis on a 2% agarose gel, in the presence of ethidium bromide. The endonuclease MunI recognizes and cleaves the sequence CA*ATTG, which occurs only once in each of the analyzed fragments. The mutations in the sixth exon of the NBN gene (c.657del5 and p.R215W) were assessed by the PCR single-strand conformation polymorphism (SSCP) method. PCR reactions were performed in a total volume of 25 µl containing 2.5 µl of 10X PCR buffer with 15 mM MgCl2, 6 pM of each primer, 2 mM of each dNTP, 1.5 units of Taq DNA polymerase and 1.5 µl of DNA template. Amplification conditions involved initial denaturation for 3 min at 95°C, followed by 5 cycles of 25 sec at 95°C, 25 sec at 50°C and 55 sec at 72°C; then 30 cycles of 25 sec at 94°C, 35 sec at 48°C, 45 sec at 72°C, with a 6-min final extension at 72°C. Next, 4 µl of the PCR products were mixed with 9 µl of the loading buffer, and denatured for 5 min at 95°C, cooled and separated on 7% non-denaturing polyacrylamide gel for 40 h 4°C. Those samples exhibiting positive results were subsequently sequenced using the Sanger method. The conditions of PCR before sequencing were the same as those before RFLP analysis for p.I171V and SSCP analysis for c.657-661del and p.R214W. PCR products were cleaned up using NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel, Inc., Düren, Germany) according to the manufacturer's protocol. The sequencing reaction protocol included 45 cycles of 10 sec at 94°C, 5 sec at 52°C and 3 min at 60°C. The sequences of all the primers used for PCR analysis in the present study are provided in Table I.

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