“Conventional plasmid based library construction using the AT-rich gDNA from Flavobacterium columnare resulted in inefficient cloning and short inserts. In contrast, we were able to construct three high quality genomic libraries (3-6 kb, 6-10 kb, and 10-12 kb) on our first attempt by using the BigEasy Linear Cloning System. At the end of shotgun sequencing, average insert sizes were as we predicted and the genome assembled nicely.”

- Dr. Attila Karsi, Mississippi State Univ.


“Using conventional cloning strategies, we have found that the large cDNAs (~14 kb) we are studying rearrange very frequently. Other systems reported to decrease rearrangement, including genetically modified E. coli, low temperature incubation, and low copy number plasmids yielded only a small improvement in the tendency of circular plasmids bearing these cDNAs to lose non-essential sequences. The results we obtained with the pJAZZ system were dramatically different: the large fragments could be stably cloned, reliably prepared in large quantity, and sequenced with great efficiency. The pJAZZ system allowed us to move forward on a technical point that had frustrated us for over a year.”


- Dr. Nicholas Sibinga, Albert Einstein College of Medicine, Dept. of Developmental & Molecular Biology
Related Products

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BigEasy® v2.0 Linear Cloning System (pJAZZ® Vectors)

  • Maximum insert stability.
  • Efficiently clone any insert up to 30 kb.
  • Create libraries from A/T-rich or G/C-rich genomes.
  • Clone gene clusters or operons.
  • Inducible copy number.

Ordering

Product Description Size Cat. No. List Price Quantity BUY
BigEasy v2.0 Linear Cloning Kit (pJAZZ-OC NotI Vector)  5 rxns 43024-1 $283.00
   10 rxns 43024-2 $482.00
   20 rxns 43024-3 $863.00
BigEasy v2.0 Linear Cloning Kit (pJAZZ-OK Blunt Vector)  5 rxns 43036-1 $283.00
   10 rxns 43036-2 $482.00
   20 rxns 43036-3 $863.00
BigEasy v2.0 Linear Cloning Kit (pJAZZ-OK NotI Vector)  5 rxns 43042-1 $283.00
   10 rxns 43042-2 $482.00
   20 rxns 43042-3 $863.00
BigEasy® v2.0 Linear Cloning Kit (pJAZZ-OC Blunt Vector)  5 rxns 43018-1 $283.00
   10 rxns 43018-2 $482.00
   20 rxns 43018-3 $863.00
BigEasy-TSA Electrocompetent Cells  6 rxns (SOLOs) 60224-1 $142.00
   12 rxns (SOLOs) 60224-2 $241.00
   24 rxns (SOLOs) 60224-3 $443.00
ORDER INFORMATION
BigEasy v2.0 Linear Cloning Kits include:  Dephosphorylated pJAZZ vector pre-cut at either a SmaI (blunt) or NotI site, CloneSmart DNA Ligase, CloneDirect™ 10X Ligation Buffer (includes ATP), Sequencing Primers, Positive Control Insert DNA, DNA Terminator® End Repair Enzyme, BigEasy-TSA Electrocompetent Cells in SOLO packaging (1 transformation per tube), Recovery Medium, Transformation Control DNA, and complete protocols. BigEasy-TSA Electrocompetent Cells are also available separately.

Description

The BigEasy Kit with the pJAZZ vector is ideal for constructing bias-free, large-insert genomic libraries, or for cloning difficult DNA of any size up to 30 kb.  Because the novel pJAZZ vector is maintained as a linear molecule, 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-rich 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 × 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.

 

pJAZZ-OC and pJAZZ-OK vectors

Figure 1. pJAZZ-OC and pJAZZ-OK linear vectors. RepA, replication factor and low copy origin of replication (~2-4 per cell; inducible 5-10 fold); Camr - chloramphenicol resistance gene; Kanr - kanamycin resistant gene; telN - protelomerase gene; cB - replication regulator. Approximate positions of transcription terminators (T) are indicated.

Figure 2. Sequence trace of a Piromyces clone, showing extremely high (96%) AT content (see figure 3d).

Figure 3. 15-20 kb PCR amplification products cloned into the pJAZZ® linear vector.

 

Figure 4. Mouse genomic DNA sheared to
6-20 kb and cloned into the pJAZZ linear vector.

 

Figure 5. Oxytricha trifallax genomic DNA (75-85% AT) sheared to 6-20 kb and cloned into the pJAZZ linear vector.

Figure 6. Piromyces genomic DNA (85% AT) cloned in the pJAZZ vector. This DNA was unclonable in all other vectors.

Convenient Success

Efficiently clone the most difficult DNAs

The BigEasy v2.0 Kits eliminate tedious vector and competent cell preparation, as well as time-consuming QC testing.  Kits include optimized reagents, ligation-ready vector, highly efficient electrocompetent cells, detailed instructions, and trouble-shooting guides.

Published Paper:

Godiska R, Mead DA, Dhodda V, Hochstein R, Karsi A, Usdin K, Entezam A, Ravin N. (2010) Linear plasmid for cloning large or repetitive sequences in E. coli. Nuc. Acids Res. 38:e88. Read Paper >>

 

 

Resources

Document File Name Type
Manual MA033-BigEasy-v20-Linear-Cloning-Kit PDF
Manual MA042-BigEasy-TSA-Electrocompetent-Cells PDF
Product Flyer BigEasy-2011 PDF
eLucidations Genomic_libraries_BigEasyv2 PDF
eLucidations eLucidations_v7 PDF
Citations BigEasy® v2.0 Linear Cloning System (pJAZZ® Vectors) - Citations LINK
Vector Sequence Sequence(s) Link
PDF MSDS PDF

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