Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
Article Title: Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes
Figure Lengend Snippet: Gateway® BP and LR reactions. BP and LR Clonase® facilitate the recombination between att B and att P and between att L and att R, generating att L and att B sequences, respectively.
Article Snippet: 150 ng/µl entry vector (e.g., pDONR™221; Life Technologies) Gateway® BP Clonase® II enzyme mix (Life Technologies) 10 to 50 ng/µl att B-PCR product prepared according to the Gateway® scheme (Basic Protocol 1) PCR-grade H2 O LB plates supplemented with 50 µg/ml kanamycin (or other appropriate antibiotic; unit 1.1 ) LB medium supplemented with 50 µg/ml kanamycin (or other appropriate antibiotic; unit 1.1 ) 50% (v/v) glycerol, sterile Plasmid DNA isolation kit (e.g., Qiagen; optional) 96-well plate or 1.5 ml microcentrifuge tubes Multichannel pipettor 25°C incubator Additional reagents and equipment for growth of T1 phage resistant (T1R ) E. coli competent cells (e.g. One Shot® MAX Efficiency® DH5α™ competent cells; Life Technologies) ( unit 1.8 ), DNA miniprep ( unit 1.6 or commercial DNA prep kit), and DNA sequencing (Chapter 7).