COMPETENT CELLS Cloning & Protein Expression

CLONING & MAPPING

DNA AMPLIFICATION & PCR
RNA ANALYSIS
PROTEIN EXPRESSION & ANALYSIS
GENE EXPRESSION & CELLULAR ANALYSIS
 CLONING & PROTEIN EXPRESSION

CLONING & MAPPING

Strain Properties DNA AMPLIFICATION & PCR

RNA ANALYSIS
There are many properties to consider when choosing a strain for your
PROTEIN EXPRESSION & ANALYSIS
experiments. Requirements such as high-quality plasmid preparations,
blue/white screening, cytoplasmic disulfide bond formation, regulated GENE EXPRESSION & CELLULAR ANALYSIS
protein expression and fast colony growth necessitate specific strain choices.
The following selection chart highlights the characteristics of NEB’s strains
to help select the optimal strain for a particular experiment.

Cloning Strain Properties

Colonies
Transformation T1 Blue/ visible Cytoplasmic
STRAIN efficiency Phage White after EndoI Protease T7 RNA disulfide bond Drug
PROPERTIES (cfu/µg)(1) Strain resistant screening lacI q lysY 6.5 hrs. deficient(2) deficient(3) F´ RecA¯ Polymerase formation(4) Resistance(5)
NEB Turbo 1–3 x 109 K12 l l l – l l – l – – – nit

NEB 5-alpha 1–3 x 10 9

K12 l l – – – l – – l – – none

NEB 5-alpha F´ I q
1–3 x 10 9
K12 l l l – – l – l l – – tet

NEB 10-beta 1–3 x 10 9

K12 l l – – – l – – l – – str
–
dam /dcm –
1–3 x 10 6
K12 l – – – – l – – – – – cam, str, nit

Protein Expression Strain Properties

NEB Express 0.6–1 x 109 B l – – – – l l – – – – nit

NEB Express I q
0.6–1 x 10 9
B l – l – – l l – – – – cam, nit

T7 Express 0.6–1 x 10 9
B l – – – – l l – – l – nit

T7 Express I q
0.6–1 x 10 9
B l – l – – l l – – l – cam, nit

T7 Express lysY 0.6–1 x 10 9

B l – – l – l l – – l – cam, nit

T7 Express lysY/I q
0.6–1 x 10 9
B l – l l – l l – – l – cam, nit

T7 Express Crystal 0.6–1 x 10 9

B l – – – – l l – – l – nit

SHuffle ™
1 x 10 6
K12 l – l – – – – l – – l str, spec

SHuffle T7 ™
1 x 10 6
K12 l – l – – – – l – l l str, spec
cam, str,
SHuffle™ T7 lysY 1 x 106 K12 l – l l – – – – – l l
spec
SHuffle™ Express 1 x 107 B l – l – – l l – – – l spec
SHuffle™
1 x 107 B l – l – – l l – – l l spec(6)
T7 Express
SHuffle™ T7
1 x 107 B l – l l – l l – – l l cam, spec(6)
Express lysY
BL21(DE3) 1–5 x 107 B l – – – – – l – – l – –

Lemo21(DE3) 1–3 x 10 7
B l – – l – – l – – l – cam
(1) TE are given high-quality for high efficiency chemically competent strains. TE for electrocompetent strains are 1-4 x 1010 cfu/µg. TE for subcloning strains are >1 x 106 cfu/µg.
(2) Important for high-quality plasmid preparation.
(3) Lacks Lon and OmpT protease activity.
(4) Constitutively expresses a chromosomal copy of the disulfide bond isomerase DsbC.
(5) nit = nitrofurantoin, tet = tetracycline, cam = chloramphenicol, str = streptomycin, spec = spectinomycin
(6) Resistance to low levels of streptomycin may be observed.

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Convenient Formats Visit the technical reference section
of www.neb.com for more
information, including tips for
NEB competent cell strains are available in various formats for your convenience. enhancing transformation efficiencies.
All are available as 50 µl single-use transformation tubes and many are offered in larger,
200 µl tubes for multiple simultaneous reactions. Our most popular cloning strains are
available as electrocompetent cells. SOC outgrowth media and control plasmids are
provided with many of these formats. For even greater value, NEB 5-alpha is also avail-
able in a lower efficiency, subcloning format.

Cloning Formats

SOC & Control

Plasmid
FORMATS Single-use 200 µl tubes Electrocompetent Subcloning Included
NEB Turbo l l l l

NEB 5-alpha l l l l l

NEB 5-alpha F´ I q l l l

NEB 10-beta l l l l

– –
dam /dcm l l l

Protein Expression Formats

NEB Express l l l

NEB Express I q l l l

T7 Express l l l

T7 Express I q l l l

T7 Express lysY l l l

T7 Express lysY/I q l l l

T7 Express Crystal l l l

SHuffle™ l

SHuffle™ T7 l

SHuffle™ T7 lysY l

SHuffle™ Express l

SHuffle™ l
T7 Express
SHuffle™ T7 l
Express lysY
BL21(DE3) l l

Lemo21(DE3) l

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 CLONING & PROTEIN EXPRESSION

Cloning Strains
NEB’s growing line of competent cells includes several popular strains for cloning and Advantages:
protein expression, in addition to strains with unique properties including fast colony
• Specialized cloning applications
growth, tight control of expression and disulfide bond formation. Our cloning strains
include derivatives of the industry standards, DH5a™ and DH10B™. NEB Turbo is • Strains for cloning toxic genes
unique to NEB and allows colony growth after 6.5 hours. NEB’s dam–/dcm– strain • Free of animal products
enables Dam and Dcm methylation free plasmid growth. Our cells are all extensively • T1 phage resistance (fhuA2)
tested for phage resistance, antibiotic resistance and sensitivity, blue/white screening and
• Media and control plasmid included
transformation efficiency. High efficiency, 5 Minute and Electroporation Protocols
are provided when applicable. • A variety of convenient formats
• Bulk sales available for
Cloning custom packaging
CHARACTERISTICS STRAIN

• Fastest growth – colonies visible after 6.5 hours

• Plasmid preparation after 4 hours of culture outgrowth NEB Turbo Competent E. coli
• Tight control/expression of toxic genes

• Versatile cloning strain

NEB 5-alpha Competent E. coli
• DH5a derivative

• Toxic gene cloning

NEB 5-alpha F´ I q Competent E. coli
• F´ strain with extremely high transformation efficiency

• Large plasmid and BAC cloning

NEB 10-beta Competent E. coli
• DH10B derivative

• Plasmid isolates lack Dam/Dcm methylation dam – /dcm – Competent E. coli

Choose NEB Turbo for Fast Colony Growth

8 hours 10 hours 12 hours

With NEB Turbo, colonies are visible after only 8 hours. Ligation products were transformed into 50 µl of NEB Turbo Competent E. coli and
plated on LB/Amp. Plates were incubated for 8 hours, 10 hours and 12 hours at 37°C. NEB Turbo features fast colony growth and blue/white
selection to simplify cloning experiments.

4
Protein Expression Strains
NEB also offers a wide variety of competent cell strains ideal for many protein expres- Advantages:
sion applications. These strains address the needs of protein expression control, toxic
• Deficient in proteases Lon and OmpT
protein expression, cytoplasmic disulfide bond formation, difficult targets and crystal-
lography. NEB Express, T7 Express and SHuffle™ strains are available with varying • Do not restrict methylated DNA
levels of control. I q strains feature the added control of IPTG-induced expression of • Free of animal products
non-T7 plasmids by lacI q . Only NEB offers the exceptional control of expression from
• T1 phage resistance ( fhuA2 )
the lysY gene that reduces basal expression from T7 strains without inhibiting IPTG-
induced expression. Our Lemo21(DE3) strain features tunable T7 expression for • Media and control plasmid included
difficult targets such as membrane proteins and proteins prone to insoluble expression. • A variety of convenient formats
Each strain is provided with a detailed protocol for optimal expression.
• Bulk sales available for
custom packaging

Protein Expression
CHARACTERISTICS STRAIN

PROTEASE DEFICIENT B STRAINS

• T7 Expression BL21(DE3)

• Tunable T7 Expression
• Expression of difficult targets including membrane proteins, Lemo21(DE3)
toxic proteins and proteins prone to insoluble expression

• Versatile non-T7 expression strain NEB Express Competent E. coli

• Control of IPTG induced expression from Plac  , Ptac and Ptrc NEB Express I q Competent E. coli

• Most popular T7 expression strain T7 Express Competent E. coli

• T7 expression
T7 Express I q Competent E. coli
• Reduced basal expression
• T7 expression
T7 Express lysY Competent E. coli
• Better reduction of basal expression

• T7 expression
• Lowest basal expression
T7 Express lysY/I q Competent E. coli Not sure which strain
• T7 expression
T7 Express Crystal Competent E. coli
to use? – Try one of
• SeMet labeling of protein for crystallography

• Enhanced capability to correctly fold proteins with multiple

our sampler packs
SHuffle™ Express Competent E. coli
disulfide bonds in the cytoplasm
Find the optimal level of expression
• T7 Expression for your experiments by purchas-
• Enhanced capability to correctly fold proteins with multiple SHuffle™ T7 Express Competent E. coli
disulfide bonds in the cytoplasm ing one of our sampler packs. The
• T7 Expression T7 express High Efficiency Sampler
• Tightly controlled expression of toxic proteins
• Enhanced capability to correctly fold proteins with multiple
SHuffle™ T7 Express lysY Competent E. coli contains 2 (200 µl ea.) tubes of
disulfide bonds in the cytoplasm each of our superior high efficiency
K12 STRAINS T7 Expression strains. The SHuffle
• Enhanced capability to correctly fold proteins with multiple Sampler Pack contains 200 µl
SHuffle™ Competent E. coli
disulfide bonds in the cytoplasm
tubes of each of the SHuffle strains.
• T7 Expression Sampler packs offer exceptional value
• Enhanced capability to correctly fold proteins with multiple SHuffle™ T7 Competent E. coli
disulfide bonds in the cytoplasm when compared to purchasing each
• T7 expression strain separately.
• Tight control/expression of toxic proteins
SHuffle™ T7 lysY Competent E. coli
• Enhanced capability to correctly fold proteins with multiple
disulfide bonds in the cytoplasm

www.neb.com 5
 ADDITIONAL
CLONING CONVENIENCE
& PROTEIN EXPRESSION
Protein Expression Strains for Difficult Targets
Expression of Toxic Proteins
T7 Expression of recombinant protein is
often improved by the co-expression of
T7 lysozyme which binds and inhibits T7
RNA polymerase function until the point
of induction. NEB has constructed unique
T7 Express derivatives with a single copy
of a T7 lysozyme gene (lysY), a single
copy of the lacI q gene or a single copy
of both genes on a mini-F plasmid, which
is maintained without antibiotic selec-
tion. This enhancement is unique to NEB
strains and makes them less susceptible to
lysis during the overexpression of an inner
membrane protein. This ensures complete
repression of T7 expression in the
absence of inducer molecule. Yet, T7
expression is activated within 30 minutes
after induction. Our lysY strains offer
maximal control of T7-mediated toxic
protein expression.
Improved transformation of toxic clones with
T7 Expression strains from NEB
60
Percent Transformed (%)
40
20
0 q
BL21 (DE3) T7 Express lysY
Transformed Strain
A T7 expression plasmid and the same plasmid containing a gene
encoding a toxic mammalian protein were transformed into each
host. Comparison of the relative transformation efficiencies demon-
strates that the T7 Express hosts provide the levels of control
necessary for transformation of potentially toxic clones. BL21(DE3)
could not be transformed with the toxic clone.
6
Proteins with Multiple Disulfide Bonds Difficult to Express Proteins
SHuffle strains from NEB are engineered Lemo21(DE3) Competent E. coli is a tun-
E. coli strains capable of expressing able T7 expression strain designed for
proteins with increased disulfide bond the expression of challenging proteins. A
complexity in the cytoplasm. SHuffle derivative of BL21(DE3), Lemo21(DE3)
strains express the disulfide bond isom- offers the host features of this popular
erase DsbC within the cytoplasm. DsbC expression strain, with the added benefit
isomerizes mis-oxidized substrates into of being able to control expression lev-
their correctly folded state greatly enhanc- els by varying the level of T7 lysozyme
ing the fidelity of disulfide bond forma- (lysY), the natural inhibitor of T7 RNA
tion. Cytoplasmic expression also results Polymerase. The fine control of expression
in significantly higher protein yields of makes Lemo21(DE3) ideal for membrane
disulfide bonded proteins when compared proteins, toxic proteins, secreted proteins
to periplasmic expression. SHuffle strains and proteins prone to insoluble expres-
are sensitive to kan, amp, tet and in most sion.
cases, cam which makes them able to
express proteins from a wide variety of
expression vectors offering greater versa-
tility in experimental design.
PfCHT1 chitinase activity assayed Overnight expression of a
from crude lysates membrane protein – PhoA fusion
10,000
6,000
8,000 5,000
4,000
Relative Activity
6,000
PhoA Activity (units)
3,000
4,000
2,000
2,000
1,000
0
q Origami (DE3) SHuffle T7 SHuffle T7 0
lysY/l l 0 100 500 1,000
Express
µM rhamnose
Plasmodium falciparum chitinase (PfCHT1) with three cysteines was Lemo21(DE3) System™ enables simple, rapid optimization of mem-
expressed from a plasmid under the regulation of a T7 promoter. After brane protein expression.
induction, cells were harvested and crude cell lysates were prepared.
PfCHT1 was assayed using a chromogenic substrate (CalBioChem
#474550) and standardized to protein concentration using Bradford
reagent.
 ORDERING INFORMATION

Cloning Strains
PRODUCT NEB # SIZE
NEB Turbo Competent E. coli C2984H 20 x 0.05 ml
NEB Turbo Competent E. coli C2984I 6 x 0.2 ml
NEB Turbo Electrocompetent E. coli C2986K 6 x 0.1 ml
NEB 10-beta Competent E. coli C3019H 20 x 0.05 ml
NEB 10-beta Competent E. coli C3019I 6 x 0.2 ml
NEB 10-beta Electrocompetent E. coli C3020K 6 x 0.1 ml
NEB 5-alpha Competent E. coli C2987H 20 x 0.05 ml
NEB 5-alpha Competent E. coli C2987I 6 x 0.2 ml
NEB 5-alpha Competent E. coli (Subcloning Efficiency) C2988J 6 x 0.4 ml
NEB 5-alpha Electrocompetent E. coli C2989K 6 x 0.1 ml
NEB 5-alpha F´ I q Competent E. coli C2992H 20 x 0.05 ml
NEB 5-alpha F´ I Competent E. coli
q
C2992I 6 x 0.2 ml
dam – /dcm – Competent E. coli C2925H 20 x 0.05 ml
dam – /dcm – Competent E. coli C2925I 6 x 0.2 ml
Component Sold Separately: SOC Outgrowth Medium B9020S 4 x 25 ml

Protein Expression Strains

PRODUCT NEB # SIZE
NEB Express Competent E. coli C2523H 20 x 0.05 ml
NEB Express Competent E. coli C2523I 6 x 0.2 ml
NEB Express I q Competent E. coli C3037H 20 x 0.05 ml
NEB Express I q Competent E. coli C3037I 6 x 0.2 ml
T7 Express Competent E. coli C2566H 20 x 0.05 ml
T7 Express Competent E. coli C2566I 6 x 0.2 ml
T7 Express I q Competent E. coli C3016H 20 x 0.05 ml
T7 Express I Competent E. coli
q
C3016I 6 x 0.2 ml
T7 Express lysY Competent E. coli C3010H 20 x 0.05 ml
T7 Express lysY Competent E. coli C3010I 6 x 0.2 ml
T7 Express lysY/I q Competent E. coli C3013H 20 x 0.05 ml
T7 Express lysY/I Competent E. coli
q
C3013I 6 x 0.2 ml
T7 Express Crystal Competent E. coli C3022H 20 x 0.05 ml
T7 Express Crystal Competent E. coli C3022I 6 x 0.2 ml
T7 Express High Efficiency Sampler C3009I 8 x 0.2 ml
SHuffle™ Competent E. coli C3025H 6 x 0.05 ml
SHuffle™ T7 Competent E. coli C3026H 6 x 0.05 ml
SHuffle™ T7 lysY Competent E. coli C3027H 6 x 0.05 ml
SHuffle Express Competent E. coli
™
C3028H 6 x 0.05 ml
SHuffle™ T7 Express Competent E. coli C3029H 6 x 0.05 ml
SHuffle T7 Express lysY Competent E. coli
™
C3030H 6 x 0.05 ml
SHuffle™ Sampler Pack C3032I 1 set
BL21(DE3) Competent E. coli C2527H 20 x 0.05 ml
BL21(DE3) Competent E. coli C2527I 6 x 0.2 ml
Lemo21(DE3) Competent E. coli C2528H 6 x 0.05 ml
Component Sold Separately: SOC Outgrowth Medium B9020S 4 x 25 ml
Note: Store Competent Cells at –80°C. Once thawed, do not refreeze. 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.

DH5α™ and DH10B™ are trademarks of Invitrogen Corporation.

For licensing information, visit www.neb.com.
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