Collection: Cloning & Competent Cells

Both join fragments through overlapping ends, but In-Fusion works with a single enzyme carrying 3'-to-5' exonuclease activity instead of the three-enzyme exonuclease, polymerase and ligase mix used in Gibson chemistry. In-Fusion needs only 15 bp of overlap where Gibson protocols typically call for 25 bp, and the reaction runs in about 15 minutes; remaining nicks are repaired by the bacteria after transformation.

Chemically competent cells are transformed by heat shock and need no special equipment, which suits routine subcloning, ligation reactions and plasmid propagation. Electrocompetent cells require an electroporator but reach far higher efficiencies — roughly 5 x 10^9 to 2 x 10^10 cfu/µg, against up to about 2 x 10^9 cfu/µg for heat shock — so they are the choice for library construction and rare-event cloning.

Recombination-prone constructs — inverted repeats, retroviral and lentiviral backbones, CRISPR gRNA libraries — are usually propagated in Endura cells, selected specifically to suppress unwanted recombination events. Where the problem is a toxic gene product rather than the sequence, OverExpress C41(DE3) and C43(DE3) carry mutations that prevent the cell death seen when such proteins are expressed in the parental BL21(DE3).

Only in a strain that lacks the methylation-restriction systems. Stellar Competent Cells (E. coli HST08) are deleted for mcrA and the mrr–hsdRMS–mcrBC region, so methylated inserts are not cleaved on entry. They are also an F– strain, which permits BAC and fosmid vectors, and they support blue/white screening with X-gal for pUC-type plasmids.

T-vectors such as pMD20 and pMD19 arrive linearized with single 3'-thymidine overhangs, so an A-tailed PCR product from a standard Taq reaction ligates straight in — quick and inexpensive when the junction sequence is unimportant. Seamless assembly earns its cost when the construct has to be scarless, directional, or built from several fragments in one reaction.

Cloning reagents from Takara Bio and competent cells from LGC Biosearch Technologies reach Israeli labs through Danyel Biotech, official Israeli distributor for both, with cold-chain delivery and hands-on help picking a strain. Request a quote on this page or send us your catalog numbers.