RsaI NEB#R0167

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	Prior to the RFLP, the multiplex PCR product was separated into three tubes for the genotyping of CYP3A4*4, CYP3A4*18B and CYP3A4*22 using BsmAI (NEB® Inc, Massachusetts, USA), RsaI (NEB® Inc, Massachusetts, USA) and BseYI (NEB® Inc, Massachusetts, USA) restriction enzymes (RE), respectively. The first tube contained 2.0 U BsmAI, 1× CutSmart NEBuffer® (NEB® Inc, Massachusetts, USA), 0.3–0.4 μg of PCR products and 9.2 μL doubly distilled water followed by incubation at 55 °C for 60 min. (Alpha Innotech, USA). The second tube contained 4.0 U of RsaI, 1× CutSmart NEBuffer® (NEB® Inc, Massachusetts, USA), 0.3–0.4 μg fresh PCR products and 8.8 doubly distilled water, followed by incubation at 37 °C for 60 min. The third tube contained 6.0 U BseYI, 1× NEBuffer 3.1® (NEB® Inc, Massachusetts, USA), 0.3–0.4 μg fresh PCR template and 8.4 μL doubly distilled water; incubated at 37 °C for 60 min followed by an inactivation step at 80 °C for 20 min. The incubation for all RFLP samples was carried out using an Accublock Digital Dry Bath.

Protocol tips

Prior to the RFLP, the multiplex PCR product was separated into three tubes for the genotyping of CYP3A4*4, CYP3A4*18B and CYP3A4*22 using BsmAI (NEB® Inc, Massachusetts, USA), RsaI (NEB® Inc, Massachusetts, USA) and BseYI (NEB® Inc, Massachusetts, USA) restriction enzymes (RE), respectively. The first tube contained 2.0 U BsmAI, 1× CutSmart NEBuffer® (NEB® Inc, Massachusetts, USA), 0.3–0.4 μg of PCR products and 9.2 μL doubly distilled water followed by incubation at 55 °C for 60 min. (Alpha Innotech, USA). The second tube contained 4.0 U of RsaI, 1× CutSmart NEBuffer® (NEB® Inc, Massachusetts, USA), 0.3–0.4 μg fresh PCR products and 8.8 doubly distilled water, followed by incubation at 37 °C for 60 min. The third tube contained 6.0 U BseYI, 1× NEBuffer 3.1® (NEB® Inc, Massachusetts, USA), 0.3–0.4 μg fresh PCR template and 8.4 μL doubly distilled water; incubated at 37 °C for 60 min followed by an inactivation step at 80 °C for 20 min. The incubation for all RFLP samples was carried out using an Accublock Digital Dry Bath.

Publication protocol

Prior to the RFLP, the multiplex PCR product was separated into three tubes for the genotyping of CYP3A4*4, CYP3A4*18B and CYP3A4*22 using BsmAI (NEB® Inc, Massachusetts, USA), RsaI (NEB® Inc, Massachusetts, USA) and BseYI (NEB® Inc, Massachusetts, USA) restriction enzymes (RE), respectively. The first tube contained 2.0 U BsmAI, 1× CutSmart NEBuffer® (NEB® Inc, Massachusetts, USA), 0.3–0.4 μg of PCR products and 9.2 μL doubly distilled water followed by incubation at 55 °C for 60 min. (Alpha Innotech, USA). The second tube contained 4.0 U of RsaI, 1× CutSmart NEBuffer® (NEB® Inc, Massachusetts, USA), 0.3–0.4 μg fresh PCR products and 8.8 doubly distilled water, followed by incubation at 37 °C for 60 min. The third tube contained 6.0 U BseYI, 1× NEBuffer 3.1® (NEB® Inc, Massachusetts, USA), 0.3–0.4 μg fresh PCR template and 8.4 μL doubly distilled water; incubated at 37 °C for 60 min followed by an inactivation step at 80 °C for 20 min. The incubation for all RFLP samples was carried out using an Accublock Digital Dry Bath.

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