Production of rProtein in E. Coli - Revision history

Raymond RedCorn at 16:11, 24 January 2019

2019-01-24T16:11:02Z

Raymond RedCorn: Raymond RedCorn moved page Production of rProtien in E. Coli to Production of rProtein in E. Coli

2019-01-16T03:59:13Z

Raymond RedCorn moved page Production of rProtien in E. Coli to Production of rProtein in E. Coli

Raymond RedCorn: Created page with "Protein Expression Using BL21(DE3) (C2527) according to New England Biolabs Inc. {{Cite web|url=https://www.neb.com/protocols/1/01/01/protein-expression-using-bl21de3-c25..."

2019-01-14T17:00:08Z

Created page with "Protein Expression Using BL21(DE3) (C2527) according to New England Biolabs Inc. <ref>{{Cite web|url=https://www.neb.com/protocols/1/01/01/protein-expression-using-bl21de3-c25..."

New page

Protein Expression Using BL21(DE3) (C2527) according to New England Biolabs Inc. <ref>{{Cite web|url=https://www.neb.com/protocols/1/01/01/protein-expression-using-bl21de3-c2527|title=Protein Expression Using BL21|last=|first=|date=|website=New England Biolabs|archive-url=|archive-date=|dead-url=|access-date=}}</ref>
# Transform expression plasmid into BL21(DE3). Plate on antibiotic selection plates and incubate overnight at 37°C.
# Resuspend a single colony in 10 ml liquid culture with antibiotic.
# Incubate at 37°C until OD600 reaches 0.4–0.8.
# Induce with 4 or 40 µl of a 100 mM stock of IPTG (final concentration of 40 or 400 µM) and induce for 3 to 5 hours at 37°C.
# Check for expression either by Coomassie stained protein gel, Western Blot or activity assay. Check expression in both the total cell extract (soluble + insoluble) and the soluble fraction only. *If a fraction of the target protein is insoluble, repeat expression at a lower temperature (15 to 30°C) or test expression in Lemo21(DE3) (NEB #C2528).
# For large scale, inoculate 1 L of liquid medium (with antibiotic) with a freshly grown colony or 10 ml of freshly grown culture. Incubate at 37°C until OD600 reaches 0.4–0.8. Add 40 or 400 µM IPTG and express protein using optimal time/temperature determined in a small scale trial.

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	Protein Expression Using BL21(DE3) (C2527) according to New England Biolabs Inc. <ref>{{Cite web\|url=https://www.neb.com/protocols/1/01/01/protein-expression-using-bl21de3-c2527\|title=Protein Expression Using BL21\|last=\|first=\|date=\|website=New England Biolabs\|archive-url=\|archive-date=\|dead-url=\|access-date=}}</ref>		Protein Expression Using BL21(DE3) (C2527) according to New England Biolabs Inc. <ref>{{Cite web\|url=https://www.neb.com/protocols/1/01/01/protein-expression-using-bl21de3-c2527\|title=Protein Expression Using BL21\|last=\|first=\|date=\|website=New England Biolabs\|archive-url=\|archive-date=\|dead-url=\|access-date=}}</ref>
	# Transform expression plasmid into BL21(DE3). Plate on antibiotic selection plates and incubate overnight at 37°C.		# Transform expression plasmid into BL21(DE3). Plate on antibiotic selection plates and incubate overnight at 37°C.

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