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  • FAQ: Is NEB Stable a suitable substitute for chemical competent Stbl2 or Stbl3?

    Yes, NEB Stable is recommended in all difficult cloning experiments.

    The genotype of NEB Stable is not related to the Stbl2 or Stbl3 cell line but the performance of NEB Stable is superior.

    The performance of all 3 strains was tested by cloning of a DNA fragment with 5 x 32bp repeats. The test construct is named pUC-5xREP and it was created using the Gibson Assembly method of cloning. Following transformation of the Gibson Assembly reaction into the respective strain, 5xREP insert stability was analyzed by colony PCR:

    Stbl2 colonies achieved 1-mm diameter in approximately 24 hrs at 30°C. This population of colonies all carried plasmid clones with deletions (33 of 33 colonies analyzed).

    Stbl3 colonies achieved 1-mm diameter in 18-20 hrs at 30°C. Of 33 colonies analyzed, 22 contained full-length 5xREP insert (67% correct).

    NEB Stable cells grow well at 30°C and 1-mm diameter colonies are obtained after 18-20 hrs at 30°C. 5xREP insert stability was best when using NEB Stable as the cloning strain (97% correct, 32 of 33 colonies analyzed).

    Importantly, NEB Stable carries the endA mutation (in contrast to Stbl3) so that isolated plasmids are free of Endonuclease I.