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How to use SHuffle®

Return to SHuffle Expression Strains
Please view the recent publication from NEB scientists for a detailed protocol of the use of SHuffle cells. For a description of the SHuffle strains available, please refer to the table at the bottom of this page.

SHuffle cells have been engineered from two E. coli lineages: K12 (NEB #C3026) and B (NEB #C3028, C3029 and C3030). For a detailed description of the strains, please refer to the SHuffle Strain Choice Table at the bottom of this page.

Proteins express to various degrees of success in SHuffle-K12 vs SHuffle-B (see table below). We therefore recommend the following general protocol for optimal protein expression in SHuffle cells:

  1. Test the expression of your protein at both SHuffle lineages (K12 vs B) and select the better expressing version.
  2. Test expression of protein in various temperatures. Grow cells at 30ºC for 5 hours, split cells, and shift to various temperatures. We suggest growing cells overnight for those shifted to lower temperatures (16ºC to room temperature).
  3. Should further optimization be necessary, continue optimization of inducer concentration, induction time, and various helper plasmids. Refer to our list of publications using SHuffle for further details in expression conditions.

Strain NEB # Characteristics Drug Resistance
SHuffle® Express Competent E. coli C3028
  • Protease Deficient B Strains
SHuffle® T7 Express Competent E. coli C3029
  • Protease Deficient B Strains
  • T7 Expression
Spec, Str*
SHuffle® T7 Express lysY Competent E. coli C3030
  • Protease Deficient B Strains
  • T7 Expression
  • Tight control/expression of toxic proteins
Cam, Spec, Str*
SHuffle® T7 Competent E. coli C3026
  • K12 Strains
  • T7 Expression
Str, Spec

* Resistance to low levels of Streptomycin may be observed.

Learn about SHuffle® strains for the expression of multi-disulfide bonded and difficult-to-express proteins