![]() Received: DecemAccepted: Published: June 2, 2011Ĭopyright: © 2011 Thieme et al. PLoS ONE 6(6):Įditor: Sudha Agarwal, Ohio State University, United States of America This method can also be applied to identify the flanking sequence of DNA elements such as T-DNA or transposon insertions, or be used for cloning of any PCR product with high specificity.Ĭitation: Thieme F, Engler C, Kandzia R, Marillonnet S (2011) Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes. We have tested this method, which we call quick and clean cloning (QC cloning), for cloning of the variable regions of immunoglobulins expressed in non-Hodgkin lymphoma tumor samples. ![]() coli where the annealed vector-insert complex is repaired and ligated. The reaction mix is then directly transformed into E. Cloning is performed using a one-step reaction that only requires incubation for 10 minutes at room temperature in the presence of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. Since only specific products contain this sequence, but none of the non-specific products, only specific products can be cloned. The other side of the linearized cloning vector has homology with a sequence present in the insert, but nested and non-overlapping with the gene-specific primer used for amplification. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplification of the insert. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. ![]() We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. ![]()
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