GC Cloning and Amplification (pSMART® GC Vectors): pSMART GC LK
- Clone PCR products from proofreading or non-proofreading polymerases
- Stabilize otherwise unclonable inserts.
- No lost clones or deleted sequences.
Ordering
GC Cloning & Amplification Kits contain: 4X GC Vector Premix (includes buffer, ATP, and either pSMART® GC HK or pSMARTGC LK); CloneSmart® DNA Ligase; T4 Polynucleotide Kinase; 10X Primer Kinase Buffer (containing ATP); EconoTaq™ DNA Polymerase; EconoTaq 10X Reaction Buffer (with Mg++); Control lacZ template plus PCR primers; CloneSmart Sequencing Primers; a choice of E. cloni 10G SUPREME (>4 x 1010 cfu/µg) or ELITE (>2 x 1010 cfu/µg) Electrocompetent Cells, or 10G Chemically Competent Cells (>1 x 109 cfu/µg), in SOLO packaging (one transformation per tube); Recovery Medium; and Control pUC DNA.
Description
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.
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Figure 1. GC Cloning Concept. PCR performed using EconoTaq™ or other non-proofreading DNA polymerase adds a single G overhang to the PCR products. Alternately, incubation of blunt-ended DNA with EconoTaq DNA Polymerase adds the 3´-G overhang. Ligation to the complementary C-overhang pSMARTGC Vector is fast and highly efficient. |
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Figure 2. The GC Cloning vectors. Ori, origin of replication; Kan, Kanamycin resistance gene; plac, lac promoter; lacZ, lacZ ORF; ROP, Repressor of priming. Approximate positions of T7 and SP6 promoters, and transcription terminators (T) are indicated. |
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).
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| Figure 3. GC Cloning vs. TOPO TA cloning. A) Total CFUs per ligation. Each vector was ligated to a chloramphenicol-resistant expression cassette directly from a PCR reaction using manufacturers’ protocols. B) Correct inserts per ligation. White colonies from the kanamycin plates were patched onto chloramphenicol plates to determine the number of clones with the expected CmR inserts. | |
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Figure 4. A Thermus species polA gene was amplified and cloned into either the pSMARTGC or pcrII TOPO TA vector. All GC clones contained the expected product; deletions and empty clones were common in the TOPO TA vector. |
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.
Resources
| Document | File Name | Type |
| Manual | MA034_pSMARTGCCloning&AmplificationKit_v3.0 | |
| Vector Sequence | Sequence(s) | Link |
| MSDS |
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