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  • FAQ: Can NEB 5-alpha competent E.coli (Subcloning Efficiency) (NEB #C2988J) be used for large fragment cloning?

    The transformation efficiency for C2988J is >1 X 10E6 cfu/μg pUC19, which is 1,000 fold less than other NEB high efficiency competent cells (1 X 10E9). If 50 μl of C2988J competent cells are transformed with 100 pg of pUC19 (2 μl of 50 pg/μl pUC19 control DNA [provided in NEB high efficiency competent cells]), 950 μl of LB should be added for outgrowth (LB only provides 28% transformation efficiency compared to SOC for this strain). If 100 μl is then spread onto a plate, only 2-3 colonies will be evident after overnight growth.

    We only recommend using subcloning efficiency competent cells (NEB #C2988J) for plasmid transformation or routine subcloning, such as inserting a 1kb fragment into a 2kb vector. A 0.8kb insert has successfully been cloned into pUC19 (2.7kb) with a transformation efficiency of 4.2 X 10E4 cfu/μg pUC19 vector (see Figure on the main product page).