Chemically competent E. coli cells suitable for high efficiency transformation and isolation of plasmid clones containing repeat elements.
Highlights
Recommended host strain for cloning genes into retroviral/ lentiviral vectors
Recommended for cloning of direct repeats and inverted repeat sequences
Reduced recombination of cloned DNA (recA1)
Efficient transformation of methylated DNA derived from eukaryotic sources or unmethylated DNA derived from PCR, cDNA and many other sources [mcrAΔ(mrr-hsdRMS-mcrBC)]
Activity of nonspecific endonuclease I (endA1) eliminated for highest quality plasmid preparations
Resistance to phage T1 (fhuA)
Suitable for blue/white screening with vectors capable of alpha-complementation
High quality plasmid preparations due to endA mutation
T1 phage resistance (fhuA)
Value pricing
Free of animal products
Application Features
Cloning unstable inserts
Isolating and propagating retroviral/ lentiviral clones
Compatible with NEBuilder HiFi DNA Assembly, Gibson Assembly reactions as well as ligation reactions
Figure 1: NEB Stable enables the isolation of plasmid clones containing repetitive DNA elements
Plasmid pUC-5xREP contains five 32-bp repeats, making it unstable in a recombination-proficient strain. A) NEB Stable competent cells or B) Stbl3 competent cells were transformed with 2 µl of a pUC-5xREP Gibson Assembly reaction containing 2.2 ng (0.00125 pmol) pUC19 vector and approximately 1 ng (0.0028 pmol) 5xREP insert. Transformed cultures were plated on LB plates containing 100 µg/ml ampicillin and incubated overnight at 30°C. The next day, colony PCR was performed using M13/pUC polylinker primers to analyze 5xREP insert stability. This figure shows the results of analyzing 33 independent colonies. The correct full-length amplicon is 623bp.
Figure 2: Benefit from the high transformation efficiencies of NEB stable competent E. coli
Transformation efficiencies were compared using manufacturers’ recommended protocols. Values shown are the average of triplicate experiments.
Figure 3: Effect of outgrowth medium on transformation efficiency
50 μl of NEB Stable competent E. coli was transformed with 100 pg of pUC19 control DNA following the provided High Efficiency Transformation Protocol with the exception of varying the outgrowth medium. NEB 10-beta/Stable Outgrowth Medium delivers the highest transformation efficiency.
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.
CAUTION: This product contains DMSO, a hazardous material. Review the MSDS before handling.
Transformation reactions with AmpR plasmids do not require an outgrowth period after addition of Outgrowth Medium. Plasmid selection using antibiotics other than ampicillin requires an outgrowth period of 60 minutes at 30°C before plating on selective media. 30°C or 37°C may be used for plate incubation, however 30°C is recommended as some constructs may be unstable at elevated temperatures.
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Specifications & Change Notifications
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The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
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