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NEB/Gibson Assembly? Cloning Kit/10 reactions/E5510S

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貨號(hào): E5510S

品牌: NEB

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- Gibson Assembly was developed by Dr. Daniel Gibson and his colleagues atthe J. Craig Venter Institute and licensed to NEB by Synthetic Genomics, Inc. Itallows for successful assembly of multiple DNA fragments, regardless of fragmentlength or end compatibility. It has been rapidly adopted by the syntheticbiology community due to its ease-of-use, flexibility and suitability for large DNAconstructs.Gibson Assembly efficiently joins multiple overlapping DNA fragments in asingle-tube isothermal reaction (1,2). The Gibson Assembly Master Mix includesthree different enzymatic activities that perform in a single buffer:
  - The exonuclease creates single-stranded 3´ overhangs that facilitate theannealing of fragments that share complementarity at one end (overlap region).
  - The proprietary DNA polymerase fills in gaps within each annealed fragment.
  - The DNA ligase seals nicks in the assembled DNA.
  The end result is a double-stranded fully sealed DNA molecule that can serveas template for PCR, RCA or a variety of other molecular biology applications,including direct transformation. The method has been successfully used by Gibson’sgroup and others to assemble oligonucleotides, DNA with varied overlaps(15–80 bp) and fragments hundreds of kilobases long (1–2).To help select the best DNA assembly method for your needs, please use our Synthetic Biology/DNA Assembly Selection Chart.For help designing primers, please view our primer design video.
  Overview of the Gibson Assembly CloningMethod
  Specification:
  10 μl of 2X Gibson Assembly Master Mix was incubated with 6 fragments(5 fragments of 400 bp and one of 2,780 bp, with 40 bp overlap, 0.05 pmoleach) in a final volume of 20 μl at 50°C for 60 minutes. NEB 5-alpha CompetentE. coli (NEB #C2987) were transformed with 2 μl of the master mix/fragmentmixture using the transformation protocol on page 12. Greater than 100white colonies were observed when 1/10 of the outgrowth was spread on anampicillin plate with IPTG/Xgal and incubated overnight.Overview of Gibson Assembly Cloning Kit Protocol:
  - Design primers to amplify fragments (and/or vector) with appropriateoverlaps
  - PCR amplify fragments using a high-fidelity DNA polymerase.
  - Prepare linearized vector by PCR amplification using a high-fidelity DNA polymerase or by restriction digestion.
  - Confirm and determine concentration of fragments and linearized vector using agarose gel electrophoresis, a NanoDrop™ instrument or other method.
  - Add fragments and linearized vector to Gibson Assembly Master Mixand incubate at 50°C for 15 minutes to 1 hour, depending on number offragments being assembled.
  - Transform into NEB 5-alpha Competent E. coli (provided) or use directly inother applications.
  This product is related to the following categories:
  DNA Assembly, Cloning and Mutagenesis Kits Products
  This product can be used in the following applications:
  Gibson Assembly®
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