C2523 FAQs for NEB Express Competent E. coli (High Efficiency), Protein Expression, Intl, NEB
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NEB Express Competent E. coli (High Efficiency) FAQ

Q1: Why are there no colonies or no growth in liquid culture (C2523)?
Q2: Why is there no protein visible on gel or no activity (C2523)?
Q3: Why is induced protein insoluble (C2523)?
Q4: What are the solutions/recipes (C2523)?
Q5: What are the strain properties (C2523)?
Q6: Can I store competent cells at -20C instead of -80C?
Q7: Are NEB's competent cells compatible with the "Plate and Go" protocol?
Q8: Can the NEB Express Competent E.coli (High Efficiency) be used for the expression of constructs containing a T7 promoter?
Q9: What is the difference between NEB #C2523H and NEB #C2523I?
Q10: What is the optimal heat shock time for this strain (NEB #C2523H and NEB #C2523I)?
Q11: How long should I incubate cells on ice after DNA has been added (NEB #C2523H and NEB #C2523I)?

Q1: Why are there no colonies or no growth in liquid culture (C2523)?

A1: There may be basal expression in the NEB Express host. If toxicity of the expressed protein is likely, transformation of the expression plasmid should be carried out in:

NEB Express Iq (
NEB# C3037): over-expression of the LacI repressor reduces basal expression.

Incubation at 30C or room temperature may also alleviate toxicity issues. In addition, check antibiotic concentration (test with control plasmid).


Q2: Why is there no protein visible on gel or no activity (C2523)?

A2: Check for toxicity - no protein may mean the cells have eliminated or deleted elements in the expression plasmid.

Culture cells for protein induction. Just before induction, plate a sample on duplicate plates with and without antibiotic selection. If toxicity is an issue, there will be a significant difference between the number of colonies on the plates. Fewer colonies will be seen on plates containing antibiotic (indicating that the plasmid has been lost) compared to plates without antibiotic.
If toxicity is the problem test the above Iq host to reduce basal level expression.


Q3: Why is induced protein insoluble (C2523)?

A3: Check for insolubility - this is important because high expression and high production of protein can result in the target protein becoming insoluble. Potential solutions for this are:
Induce at lower temperatures (as low as 15C overnight)
Reduce IPTG concentration to between 0.01 mM and 0.1 mM
Induce for less time (as little as 15 minutes)
Induce earlier in growth (OD600 = 0.3 or 0.4)


Q4: What are the solutions/recipes (C2523)?

A4: SOB:
2% Vegetable peptone (or Tryptone)
0.5% Yeast Extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4

SOC:
SOB + 20 mM Glucose

LB agar:
1% Tryptone
0.5% Yeast Extract
0.17 M NaCl
1.5% Agar


Q5: What are the strain properties (C2523)?

A5: The properties of this strain that contribute to its usefulness as a protein expression strain are described below. The genotypes underlying these properties appear in parentheses.

Protease Deficient ([lon] ompT): E. coli B strains are "naturally" deficient in the lon protease which in K-12 strains serves to degrade misfolded proteins and to prevent some cell cycle-specific proteins from accumulating. The OmpT protease resides at the surface of wild type E. coli in both K-12 and B strains, presumably helping the cells to derive amino acids from their external environment. Cells deficient in both these proteases are much more amenable to the production of proteins from cloned genes.

Recovery from DNA Damage (sulA11): E. coli cells can tolerate a substantial amount of chronic DNA damage as long as repair is allowed to proceed. This capacity is compromised if the cells are unable to divide following repair. In lon- cells, SulA, a cell division inhibitor, accumulates and causes cells to become hypersensitive to DNA damage. The sulA mutation introduced into the T7 Express strain allows cells to divide more normally in the absence of Lon protease.

Endonuclease I Deficient: (endA) The periplasmic space of wild type E.coli cells contains a nonspecific endonuclease. Extreme care must be taken to avoid degradation of plasmids prepared from these cells. The endA mutation deletes this endonuclease and can significantly improve the quality of plasmid preparations.

Restriction Deficient: (Δ(mcrC-mrr)114::IS10) Wild type E. coli B strains carry a Type I restriction endonuclease which cleaves DNA with the site TGA(N8)TGCT. While E. coli DNA is protected from degradation by a cognate methyl-transferase, foreign DNA will be cut at these sites. The deletion described above eliminates both the methylase and the endonuclease.

Methyl Restriction Deficient: (Δ(mcrC-mrr)114::IS10 and R(mcr-73::miniTn10--TetS)2): E. coli has a system of enzymes encoded by mcrA, mcrBC and mrr which will cleave DNA with methylation patterns found in higher eukaryotes, as well as some plant and bacterial strains. All three Mcr enzymes and Mrr have been inactivated in T7 Express allowing the introduction of eukaryotic DNA of genomic origin (e.g. primary libraries) if desired.

T1 Phage Resistant: (fhuA2) T1, an extremely virulent phage requires the E. coli ferric hydroxamate uptake receptor for infectivity. Deletion of this gene confers resistance to this type of phage, but does not significantly affect the transformation or growth characteristics of the cell.


Q6: Can I store competent cells at -20C instead of -80C?

A6: Competent cells should be stored at -80C. Storage at -20C will result in a significant decrease in transformation efficiency (TE). When tested on NEB 5-alpha Competent E.coli (NEB #C2987H), cells lost 94.5% of TE after only 24 hours ofstorage at -20C. Cells lost 98.9% of TE after 2 days, and 99.6% of TE after one week of storage at -20C.


Q7: Are NEB's competent cells compatible with the "Plate and Go" protocol?

A7: There is a "Plate and Go" protocol that provides a quick way to transform your cells by simply adding plasmid to cells and plating. No heat shock step is required. NEB has tested our and other companies' cells and all experienced a drop in efficiency. However, since NEB cells are highly efficient, the drop in efficiency may be sufficient for your needs.


Q8: Can the NEB Express Competent E.coli (High Efficiency) be used for the expression of constructs containing a T7 promoter?

A8: No. NEB Express Competent E.coli do not support the expression of constructs containing a T7 promoter. For the expression of those constructs we recommend T7 Express Competent E.coli (High Efficiency) (
NEB #C2566).


Q9: What is the difference between NEB #C2523H and NEB #C2523I?

A9: They are the same cells with the same efficiency but provided in different formats. C2523H is packaged with 20 single-use transformation tubes, each containing 50 μl of competent cells. Plasmid or ligation product can be added directly into the transformation tubes for convenience. C2523I is packaged with 6 tubes, each containing 200 μl of competent cells. The tubes should be thawed on ice and 50 μl of cells transferred into new tubes prior to transformation. Each tube contains enough cells for 4 transformations with the benefit of reducing the cost of each transformation. If you perform 3 or 4 transformations at a time, using C2523I is cost effective. Refreezing the competent cells after thawing is not recommended since it will significantly reduce transformation efficiencies.


Q10: What is the optimal heat shock time for this strain (NEB #C2523H and NEB #C2523I)?

A10: Heat shock at 42C for 20 seconds results in the highest transformation efficiency for NEB Express competent E.coli (NEB #C2523H and NEB #C2523I). Expect approximately 50% loss in transformation efficiency when heat shocking for 80 seconds (see Figure on the main product page).


Q11: How long should I incubate cells on ice after DNA has been added (NEB #C2523H and NEB #C2523I)?

A11: Incubating DNA with NEB Express competent cells on ice for 30 minutes is recommended. When tested with pUC19, an incubation time of 20 minutes did not hurt the transformation efficiency (see Figure on the main product page).