GC Cloning and Amplification (pSMART® GC Vectors): pSMART GC HK

Advanced PCR cloning
GC Cloning (patents pending) is the first major advance in PCR cloning since the first description of TA-cloning (Mead, D., et. al. (1991) Biotechnology 9, 657.) TA-cloning takes advantage of the well-known property of non-proofreading DNA polymerases (e.g., Taq, Tfl, Tth) to add a single 3´-A to PCR products.

The GC Cloning technology is based on the discovery that these same enzymes add a single 3´-G to DNA molecules, either during PCR or as a separate G-tailing reaction to any blunt DNA (Figure 1). The pSMART® GC vectors (Figure 2) contain a single 3´-C overhang, which is compatible with the single 3´-G overhang on the inserts.

Stabilized inserts
These cloning kits are based on patented pSMART® vectors (Figure 1) which are transcription- and translation-free cloning vectors designed to eliminate many of the problems associated with cloning recalcitrant DNA in conventional plasmid vectors [download 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 eLucidations™ article:  "Missing a Gene?".

Superior Performance
Compared to TOPO TA Cloning, pSMART GC Vectors produce more colonies per ligation and more colonies with the correct insert (Figure 3).  Furthermore, PCR products in pSMART GC show significantly higher stability than the same insert in a TOPO TA vector (Figure 4).

Convenient Success
Efficiently clone your PCR product regardless of its size, base composition, or enzyme used to generate it. Kits include reagents for amplification (GC Cloning and Amplification Kits, only) and ligation, 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 PCR cloning.