BglII NEB#R0144

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	The α2 gene was genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, as previously reported (41), using an MJ Mini™ Gradient Thermal Cycler (Bio-Rad, Hercules, CA, USA), followed by BglII and NdeI [New England Biolabs (NEB), Ipswich, MA, USA] restriction enzyme analysis. The PCR samples (∼1 µg) were digested overnight with BglII and NdeI (NEB) at 37°C in a temperature-controlled water bath (Bio-Rad). The digested amplicons were analyzed by electrophoresis in 2% agarose gels using ethidium bromide (Invitrogen Life Technologies) and a 2UV™ transilluminator (UVP, LLC, Upland, CA, USA).

Protocol tips

The α2 gene was genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, as previously reported (41), using an MJ Mini™ Gradient Thermal Cycler (Bio-Rad, Hercules, CA, USA), followed by BglII and NdeI [New England Biolabs (NEB), Ipswich, MA, USA] restriction enzyme analysis. The PCR samples (∼1 µg) were digested overnight with BglII and NdeI (NEB) at 37°C in a temperature-controlled water bath (Bio-Rad). The digested amplicons were analyzed by electrophoresis in 2% agarose gels using ethidium bromide (Invitrogen Life Technologies) and a 2UV™ transilluminator (UVP, LLC, Upland, CA, USA).

Publication protocol

Genotyping for the α2 gene was performed to analyze the possible genetic association between DR and the polymorphic variants of the α2 gene. The 3,160 A/G and 3,090 T/C polymorphism of the α2 intron G region were tested. The α2 gene was genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, as previously reported (41), using an MJ Mini™ Gradient Thermal Cycler (Bio-Rad, Hercules, CA, USA), followed by BglII and NdeI [New England Biolabs (NEB), Ipswich, MA, USA] restriction enzyme analysis. In brief, the method for the PCR was as follows: 250 ng genomic DNA, 0.5 µM primers (forward, 5′-GAT TTA ACT TTC CCG ACT GCC TTC-3′ and reverse, 5′-CAT AGG TTT TTG GGG AAC AGG TGG-3′), 0.2 mM deoxyribonucleotide triphosphates (Invitrogen Life Technologies, Carlsbad, CA, USA), 1.5 mM MgCl2 and 2.5 units of Taq DNA polymerase (Invitrogen Life Technologies) were combined to form the reaction mixture. The 600-bp segment of intron G encompassing the BglII and NdeI sites was amplified from genomic DNA in a final volume of 25 µl using the following conditions: 2 cycles of 94°C for 1 min, 69°C for 1 min and 72°C for 1 min; 2 cycles of 94°C for 1 min, 67°C for 1 min and 72°C for 1 min; and 30 cycles of 94°C for 1 min, 65°C for 1 min and 72°C for 1 min. The PCR samples (∼1 µg) were digested overnight with BglII and NdeI (NEB) at 37°C in a temperature-controlled water bath (Bio-Rad). The digested amplicons were analyzed by electrophoresis in 2% agarose gels using ethidium bromide (Invitrogen Life Technologies) and a 2UV™ transilluminator (UVP, LLC, Upland, CA, USA).

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