Feb 11, 2022
Version 2
Protein Expression Using BL21(DE3), Large Scale (C2527) V.2
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
Protocol Citation: New England Biolabs 2022. Protein Expression Using BL21(DE3), Large Scale (C2527). protocols.io https://dx.doi.org/10.17504/protocols.io.bhk2j4yeVersion created by Julia Rossmanith
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 17, 2020
Last Modified: February 11, 2022
Protocol Integer ID: 38266
Keywords: T7 Expression Strain, expression plasmid, transform expression plasmid into BL21(DE3)
Abstract
This is the protocol for a large-scale Protein Expression Using BL21(DE3) Competent E. coli cells (C2527).
Guidelines
BL21(DE3) Genotype:
fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS
λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5
NOTES:
1. Caution: This product contains DMSO, a hazardous material. Review the MSDS before handling.
2. Storage and Handling: Competent cells should be stored at -80°C. Storage at -20°C will result in a significant decrease in transformation efficiency. Cells lose efficiency whenever they are warmed above -80°C, even if they do not thaw.
Materials
MATERIALS
BL21(DE3) Competent E.coli - 20x0.05 mlNew England BiolabsCatalog #C2527H
BL21(DE3) Competent E.coli - 6x0.2 mlNew England BiolabsCatalog #C2527I
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Caution: This product contains DMSO, a hazardous material. Review the MSDS before handling.
Before start
Determine the optimal time/temperature for the protein expression in a small scale trial.
Transform expression plasmid into BL21(DE3).
Plate on antibiotic selection plates.
Incubate Overnight at 37 °C .
Inoculate 1 L liquid medium (with antibiotic) with a freshly grown colony or with 10 mL freshly grown culture (from a single colony) .
Incubate at 37 °C until OD600 reaches 0.4–0.8.
Induce with 4 µL or 40 µL of 100 millimolar (mM) IPTG stock (final concentration of 40 micromolar (µM) or 400 micromolar (µM) ).
Express protein using optimal time/temperature determined in a small scale trial (3 to 05:00:00 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.
Note
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).