New England Biolabs
NEB 5 alpha high efficiency competent E.coli cells
C2987I
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Routing cloning plasmid constructs of size less than 8 Kb constructed by restriction cloning were transformed into NEB 5 alpha high efficiency competent E. coli cells by heat shock method. After transformation, colonies were selected on selective agar plate and found that efficiency of transformations were 10^7 cells per microgram of plasmid construct used.
Transformation of the cloning plasmid constructs
2 microliter of plasmid constructs (200 ng) per 50 microliter of NEB 5 alpha high efficiency competent E. coli cells
I have followed the long incubation protocol which is given in the kit's instruction manual. After thawing the competent cells on ice, 2 microliter of cloning plasmid construct was added to competent cells and allowed to sit on ice for 30 minutes. Then heat shocked for 30 seconds at 42 degree C. Then cooled on ice for 5 minutes and then recovered in SOC medium at 37 degree C shaking incubator for 90 minutes. Appropriate dilutions were plated on to selective agar plates. Selective agar plates were checked on next day to estimate transformation efficiency and presence of plasmid construct by colony PCR.
Efficient thawing of competent cells and use of pre-warmed SOC medium and selective agar plates will make higher recovery of transformed cells
Overall, transformation of the cloning plasmid construct resulted in 10^7 cells per microgram of plasmid used. Presence of correct plasmid constructs were confirmed by colony PCR.
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Efficient and cheap competent cells to use. Saves lot of time and easy to follow protocol.
Highly recommended for molecular biology which routinely does large scale plasmid constructions for cloning and expression.