BfuI (BciVI) (5 U/µL)

Protocol tips

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	VL PCR products (7.0 µL per reaction) were digested with 5 units of the restriction enzyme BciVI (BfuI) (Thermo Fisher Cat# ER1501) in a 20 µL reaction at 37°C for 2 hr. The enzyme was inactivated by heating to 80°C for 20 min.

Protocol tips
VL PCR products (7.0 µL per reaction) were digested with 5 units of the restriction enzyme BciVI (BfuI) (Thermo Fisher Cat# ER1501) in a 20 µL reaction at 37°C for 2 hr. The enzyme was inactivated by heating to 80°C for 20 min.

Publication protocol

Following the cDNA synthesis reaction, we replica plated ≈5% of the volume (1.0 µL of 10-fold dilution) of the cDNA synthesis product to a third 96 well plate to serve as the template for PCR amplification of the IgG VH and VL domain sequences. As such, the templates for the VH and VL domain PCR amplification represented the cDNA yield from ≈3,000–7,500 hybridoma cells. Amplification of Ig V region sequences and fusion PCR (F-PCR) to join VL and VH PCR products were performed as described (Crosnier et al., 2010; Müller-Sienerth et al., 2014) with the noted modifications. Briefly, degenerate primer sets were used to amplify mouse Ig kappa VL and VH sequences using PFU Ultra II Fusion HS DNA polymerase (Agilent Technologies Cat# 600670) and Advantage 2 Polymerase Mix (ClonTech Cat# 639201) for VL and VH amplification, respectively. PCR conditions for VL amplification were: 95°C for 5 min; 5 cycles of 95°C for 20 s, 60°C for 20 s, 72°C for 30 s; 19 cycles of 95°C for 20 s, 60.5°C for 20 s with 0.5°C decrement per cycle, 72°C for 30 s; 10 cycles of 95°C for 20 s, 55°C for 20 s, 72°C for 30 s; 72°C for 15 min. PCR conditions for VH amplification were: 95°C for 5 min; 5 cycles of 95°C for 45 s, 62°C for 30 s, 72°C for 1 min; 19 cycles of 95°C for 45 s, 64.5°C for 30 s with 0.5°C decrement per cycle, 72°C for 1 min; 10 cycles of 95°C for 45 s, 55°C for 30 s, 72°C for 1 min; 72°C for 15 min. VL PCR products (7.0 µL per reaction) were digested with 5 units of the restriction enzyme BciVI (BfuI) (Thermo Fisher Cat# ER1501) in a 20 µL reaction at 37°C for 2 hr. The enzyme was inactivated by heating to 80°C for 20 min.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for BfuI (BciVI) (5 U/µL) below.

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