DNA Cloning and Assembly Methods pp 37-48

Part of the Methods in Molecular Biology book series (MIMB, volume 1116) | Cite as

Quick and Clean Cloning

  • Frank Thieme
  • Sylvestre Marillonnet


Identification of unknown sequences that flank known sequences of interest requires PCR amplification of DNA fragments that contain the junction between the known and unknown flanking sequences. Since amplified products often contain a mixture of specific and nonspecific products, the quick and clean (QC) cloning procedure was developed to clone specific products only. QC cloning is a ligation-independent cloning procedure that relies on the exonuclease activity of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. A specific feature of QC cloning is the use of vectors that contain a sequence called catching sequence that allows cloning specific products only. QC cloning is performed by a one-pot incubation of insert and vector in the presence of T4 DNA polymerase at room temperature for 10 min followed by direct transformation of the incubation mix in chemo-competent Escherichia coli cells.

Key words

Ligation-independent cloning Flanking sequences Specific products Genome walking Exonuclease digestion 


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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Frank Thieme
    • 1
  • Sylvestre Marillonnet
    • 2
  1. 1.Icon Genetic GmbHHalleGermany
  2. 2.Department of Cell and Metabolic BiologyLeibniz-Institut für PflanzenbiochemieHalleGermany

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