BigEasy® Long PCR Cloning Kit (pJAZZ® Vectors)

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 BigEasy Long PCR Cloning Kits contain everything needed to efficiently clone blunt or G-tailed PCR products up to 30 kb into the unbiased, high-fidelity pJAZZ linear cloning vector.  There are two different versions of the vector (Figure 2) that are compatible with either proofreading (pJAZZ-OK Blunt Vector) or non-proofreading (pJAZZ-OK GC Vector) PCR polymerases.

Stabilized inserts

The pJAZZ vector is ideal for cloning difficult PCR products of any size up to 30 kb (Figure 3).  Because the novel pJAZZ vector is linear, the ends of the vector can rotate freely.  As a result, the vector does not supercoil.  Without the torsional stress induced by supercoiling, otherwise unclonable sequences (e.g., repetitive, or A/T- or G/C-rich sequences) are stabilized. The pJAZZ linear cloning vector is maintained at low copy number (5-10/cell) in BigEasy-TSA™ Electrocompetent Cells  (>4 x 10¹⁰ cfu/µg), which are required for transformation and propagation. The copy number can be induced 5-10X in this strain. In addition, the pJAZZ vector incorporates Lucigen’s CloneSmart® technology for transcription-free cloning, which further increases insert stability.  pJAZZ clones can be isolated using conventional plasmid prep methods  and sequenced using standard techniques. See protocol for high-throughput Sanger sequencing of pJAZZ clones.

Convenient Success

Efficiently clone your PCR product regardless of its size, base composition, or enzyme used to generate it.  Kits include ligation-ready vector (no post-ligation cleanup step required), and highly efficient TSA electrocompetent cells (up to >4 X 10¹⁰ cfu/µg), detailed instructions, and trouble-shooting guides to simplify PCR cloning.