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Description
Lucigen's new CopyRight® v2.0 vectors and BAC-Optimized Replicator™ v2.0 Electrocompetent Cells combine the revolutionary, patented CloneSmart® transcription-free technology, inducible copy amplification, and a lacZ-sacB stuffer fragment that eliminates uncut vector from recombinants. The result is unprecedented accuracy, reliability, and efficiency in BAC cloning and library construction. Unlike other BAC cloning systems, Lucigen's CloneSmart technology assures that even "unclonable" inserts are stably maintained. Sequences are not lost or rearranged in the cloning process. A simple "on command" amplification system built into the CopyRight v2.0 vectors and BAC-Optimized Replicator v2.0 Electrocompetent Cells gives a 20-50 fold increase in copy number for high yields and easy purification (Wild J, et al. (2002) Genome Res. 12(9):1434-44).
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CopyRight pSMART® BAC vectors: |
The CopyRight pSMART BAC cloning vector (Figure 1) incorporates transcription-free cloning for the highest stability BAC cloning. The vector is supplied precut at a choice of BamHI, EcoRI, or HindIII sites, with dephosphorylated ends. Insert DNAs with 5’- phosphorylated ends are ligated to the CopyRight vector and transformed into Lucigen’s BACOptimized Replicator v2.0 Electrocompetent Cells.
The new pSMART BAC vectors have a lacZ-sacB stuffer region, allowing uncut vector to be detected by blue/white screening and selected against by plating on sucrose. This feature completely eliminates uncut vector background, improving the efficiency of colony picking. However, unlike other cloning vectors, the promoter and the coding sequence of the stuffer fragment are completely removed during processing. This design prevents active expression of the insert DNA by the lacZ or sacB promoter, contributing greatly to plasmid stability, especially for inserts containing toxic coding sequences, strong secondary structure, or other deleterious features.
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The CopyRight pSMART® BAC vector contains the following features: |
- Zero background
- Transcription terminators to stabilize recombinant clones
- Transcription/translation-free cloning for unstable DNAs
- lacZ/sacB stuffer that is completely removed for minimal background and no bias
- Single-copy replication origin and inducible medium-copy replication origin
- Bacteriophage lambda cos site for lambda packaging or terminase cleavage
- loxP site for Cre-recombinase recognition
- Rare-cutting restriction sites on either side of insert
- Chloramphenicol-resistance gene
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| Figure 1. Diagram of CopyRight vector pSMART BAC. ori2, repE, IncC - origin of replication (single copy); oriV - inducible origin of replication; par A,B,Cpartition genes; Cmr- chloramphenicol-resistance gene; cosN - lambda packaging signal; T – CloneSmart transcription terminators; sacB, sucrase gene; lacZ, alpha peptide portion of the beta galactosidase gene. Approximate positions of sequencing primers and transcription terminators are indicated. |
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| CopyRight pEZ™ BAC BamHI-cut or Blunt vector: The pEZ BAC vector contains the lacZ gene for blue/white screening. It is ideal for BAC cloning and library construction. The pEZ BAC vector cloning efficiency is >99%. This vector is precut at either a BamHI or HpaI blunt insertion site. |
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| Figure 2. Diagram of the CopyRight vector pEZ BAC. The pEZ BAC (for blue/white screening) vector is illustrated, showing positions of terminators (T), chloramphenicol- resistance gene (Cmr), cos site, partition genes (parA, parB, parC), single copy origin (ori2, repE), inducible origin (oriV), cloning sites, and sequencing primer sites. |
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Advantages |
- Greater cloning fidelity. Transcription-free, single copy cloning minimizes cloning bias and sequencing gaps. Clone even "unclonable" DNAs such as toxic genes, strong promoters, repeats, hairpins, and AT-rich sequences with the pSMART BAC transcription-free vector (see Figure 1).
- Maximum insert stability. CloneSmart transcription terminators in the pSMART BAC vector minimize deletions and rearrangements.
- Higher throughput. Zero background eliminates wasted time in colony picking and analysis.
- Inducible Copy Number. Increase DNA yields 20-50 fold with simple chemical induction.
- Fast and easy cloning protocol. Simply add your DNA, incubate, and transform.
- Higher cloning and sequencing efficiencies. Ultra-high efficiency BAC-Optimized Replicator v2.0 Electrocompetent Cells (>2 x 1010 cfu/μg of pUC 19) maximize recombinant yields.
- Better Value. CopyRight Kits offer superior performance for BAC cloning at a much better price than other available kits. For example, see the Random Shear BAC library generated with pSMART BAC (Figure 3).
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| Figure 3. Arabidopsis Random Shear BAC Library in the pSMART BAC vector. 7,680 clones were obtained with an average insert size of 105 kb and ~5X genome coverage. |
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| BAC-Optimized Replicator v2.0 Genotype: F¯ mcrA D(mrr-hsdRMS-mcrBC) f80dlacZDM15 DlacX74 endA1 recA1 araD139 D(ara,leu)7697 galU galK rpsL nupG (attL araC-PBAD-trfA250 bla attR) l- |
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Replicator FOS Strain |
- BAC library construction.
- General cloning of any DNA (especially inserts > 30kb).
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| Note: BAC-Optimized Replicator v2.0 Cells are required for copy number amplification. |
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