GC Cloning™ & Amplification (p

GC Cloning Technology

GC Cloning is similar to conventional TA-cloning (Mead, D., et. al. (1991) Biotechnology 9, 657), which 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 pGC™ Blue Vector contains a single 3´-C overhang, which is compatible with the single 3´-G overhang on the inserts. The pGC Blue vector includes the lacZ sequence for blue/white screening, as well as dual opposed RNA polymerase promoters for in vitrotranscription of the cloned insert (Figure 2).

Stabilized inserts

The pGC Blue Vector incorporates the transcription--free CloneSmart technology (Figure 1) to eliminate many of the problems associated with cloning recalcitrant DNA [download eLucidations™ article (PDF) and PowerPoint presentation (PPT)]. Strong transcription terminators flank the cloning site to block spurious transcription from the vector into the insert and from the insert into the vector.

Figure 1. GC Cloning. 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 pGC Blue Vector is fast and highly efficient.

Figure 2. pGC Blue Vector. 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.

Superior Performance

Lucigen GC Cloning Vectors produce more recombinants per ligation than TOPO TA Cloning (Figure 3A). In addition, compared to TOPO TA Cloning, pSMART GC Vectors produce more clones containing the correct insert (Figure 3B).

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.

Convenient Success

Efficiently clone your PCR product regardless of its size or base composition. Kits include:

• Reagents for amplification and ligation
• Ligation-ready vector (no post-ligation cleanup step required)

The GC Cloning & Amplification (pGC™ Blue) kits are optimized for use with E. cloni® 10G electrocompetent andchemically competent cells (sold separately)

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