pNIC28-Bsa4KpnI

Protocol tips

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	For the overexpression of the cloned genes, overnight LB cultures of the corresponding E. coli BL21(DE3) clones and negative-control strain E. coli BL21(DE3)/pNIC28-Bsa4KpnI were diluted to an OD620 of 0.1 in 30 ml fresh LB medium with kanamycin and further incubated at 37°C until reaching OD620 of around 0.6, and then 1 mM IPTG was added and incubation was extended for 5 additional hours.

Protocol tips
For the overexpression of the cloned genes, overnight LB cultures of the corresponding E. coli BL21(DE3) clones and negative-control strain E. coli BL21(DE3)/pNIC28-Bsa4KpnI were diluted to an OD620 of 0.1 in 30 ml fresh LB medium with kanamycin and further incubated at 37°C until reaching OD620 of around 0.6, and then 1 mM IPTG was added and incubation was extended for 5 additional hours.

Publication protocol

For the overexpression of the cloned genes, overnight LB cultures of the corresponding E. coli BL21(DE3) clones and negative-control strain E. coli BL21(DE3)/pNIC28-Bsa4KpnI were diluted to an OD620 of 0.1 in 30 ml fresh LB medium with kanamycin and further incubated at 37°C until reaching OD620 of around 0.6, and then 1 mM IPTG was added and incubation was extended for 5 additional hours. Cells were subsequently harvested and washed with sterile 0.9% NaCl and resuspended in 1 ml lysis buffer (25 mM Tris-HCl [pH 8] with NaCl 100 mM) prior to their lysis by sonication with a Branson Sonifier 150 equipped with a microprobe. Finally, lysed cells were centrifuged at 4°C and 21,130 × g for 20 min, and supernatants were collected as soluble intracellular extracts for SDS-PAGE analysis and in vitro enzyme assays. SDS-PAGE analysis of cell extracts was performed in a 4 to 20% Mini-Protean TGX precast gel (Bio-Rad, Hercules, CA, USA) using a PageRuler prestained 10- to 180-kDa protein ladder (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) as a molecular mass marker.

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein Expression Prokaryotic cells - E. coli oxidoreductases using pNIC28-Bsa4KpnI from Marie F. Gorwa-Grauslund, Applied Microbiology, Department of Ch.

Manufacturer protocol

Download the product protocol from Marie F. Gorwa-Grauslund, Applied Microbiology, Department of Ch for pNIC28-Bsa4KpnI below.

Videos

Check out videos that might be relevant for performing Protein Expression Prokaryotic cells - E. coli oxidoreductases using pNIC28-Bsa4KpnI from Marie F. Gorwa-Grauslund, Applied Microbiology, Department of Ch. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.