As much as a 500% increase in cloning efficiency
Double-stranded DNA fragments for library construction or large-scale sequencing projects are often generated by mechanically shearing larger (genomic) DNA, a process that primarily leaves uneven ends. Large DNA may also be cut with restriction enzymes to generate smaller fragments for subcloning. Sheared or restricted DNA fragments usually have one or more overhanging bases at the 5'- or 3'-termini that must be removed or “end-repaired” before ligation into blunt-end cloning vectors (e.g., Lucigen’s CloneSmart®, CopyRight®, or BigEasy® systems).
The DNATerminator Kit was developed specifically for high efficiency end repair of sheared or restriction-digested DNA.
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DNATerminator Advantages
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- Higher cloning efficiency. The DNATerminator Kit is up to five fold more efficient in generating blunt ends than commonly used procedures, such as treatment with mung bean nuclease or T4 DNA polymerase + Klenow fragment.
- Optimized system for consistent & reliable results. DNA fragmented by nebulization, sonication, hydrodynamic shearing, or nuclease treatment is efficiently rendered blunt and ligation ready.
- Maximum convenience. The Kit is ready to use and the protocol is easy to perform: no need to prepare and optimize a “homebrew” mix of enzymes and buffers.
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DNATerminator Examples |
| Figure 1 demonstrates the increase in recombinants obtained with the DNATerminator Kit compared to standard end-repair enzymes. |
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Figure 1. Effect of DNA shearing and end-repair methods on library construction efficiency. Shotgun libraries were constructed using 250 ng of lambda DNA sheared by sonication (Son) or by the GeneMachines® HydroShear™ device (HS). Sheared DNA was repaired with T4 DNA polymerase and Klenow fragment (TK) or DNATerminator End Repair Kit (DT) and cloned using Lucigen’s CloneSmart® system. Values are colony forming units (cfu) per library.
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DNATerminator Applications |
- Library construction (e.g., shotgun cloning) of fragments generated by physical shearing
- Cloning DNA generated by restriction enzymes into non-compatible sites
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DNATerminator Specifications
Each DNATerminator reaction end repairs 1 to 10 μg of Hydroshear fragmented, sonicated, nebulized, or restriction-digested DNA. These fragments can serve as inserts into any blunt cloning site. |
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