Lucigen’s unique cloning kits with pre-processed vectors and highly competent cells eliminate hours of tedious preparation and enable efficient cloning. Ligation can be completed in as little as 30 minutes with no clean-up needed; only a brief heat denaturation is necessary before transformation. Downstream manipulation (e.g, mutagenesis, subsequent insert addition) is facilitated by the minimal vector size (1.7-2.0 kb).
Stabilized Inserts and Gap-Free Cloning. All cloning kits include the transcription- and translation-free pSMART® vectors (Figure 1), which eliminate many of the problems associated with cloning recalcitrant DNA in conventional plasmid vector [see eLucidations™ article (PDF) and PowerPoint presentation (PPT)]. Conventional cloning vectors contain promoters (e.g., lacZ promoter) that constitutively transcribe the insert sequence. This transcription and coupled translation can destabilize inserts containing coding regions, strong promoters, short repeats, or incompatible secondary structures. In addition, strong transcription terminators flank the cloning site to block spurious transcription from the vector and insert-driven transcription into the vector. Low copy number versions of pSMART further stabilize sequences that are difficult to maintain in typical vectors. See "Missing a Gene?" (eLucidations Vol. 4.) [pdf].
The pSMART vectors are supplied pre-digested, with blunt, dephosphorylated ends, and are qualified to produce 99.5% recombinant clones in typical experiments. The ultra-low background of empty vector (less than 0.5%) eliminates the need to screen for recombinants, removes false negatives (light blue pUC colonies) and false positives (white colonies that lack inserts), and enables library construction from nanograms of DNA.
Figure 1. High copy (HC) and low copy (LC) versions of the pSMART transcription-free cloning vectors. Amp - ampicillin resistance gene; Kan - kanamycin resistance gene; Ori - origin of replication; ROP - "Repressor of Primer" copy number gene.
Efficiently obtain your clone or create your genomic or cDNA library—the CloneSmart, Cloning Kits eliminate tedious vector and competent cell preparation, as well as time-consuming QC testing experiments. Kits include optimized reagents, ligation-ready vector (no post-ligation cleanup step required), highly efficient electrocompetent cells (up to >4 X 1010 cfu/µg) or chemically competent cells ( >1 X109 cfu/µg), detailed instructions, and trouble-shooting guides to simplify cloning and sequencing.