Feb 11, 2022
Version 2
Protein Expression Using BL21(DE3) (C2527) V.2
- 1New England Biolabs
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Protocol Citation: New England Biolabs 2022. Protein Expression Using BL21(DE3) (C2527). protocols.io https://dx.doi.org/10.17504/protocols.io.bdd4i28wVersion created by New England Biolabs
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: March 08, 2020
Last Modified: February 11, 2022
Protocol Integer ID: 33948
Keywords: T7 Expression Strain, expression plasmid, transform expression plasmid into BL21(DE3), BL21, C2527
Abstract
This protocol explains methods for Protein Expression Using BL21(DE3) Competent E. coli (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.
Transform expression plasmid into BL21(DE3).
Plate on antibiotic selection plates.
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 µL or 40 µL of 100 millimolar (mM) IPTG stpck (final concentration of 40 or 400 µM).
Induce for 3-5 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.
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).
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 micromolar (µM) or 400 micromolar (µM) IPTG and express protein using optimal time/temperature determined in a small scale trial.