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Figure 3. Establishment of SSA in NPE. (A) Preparation of the SSA substrate pRW4′. Plasmid pRW4 was digested with XhoI and then partially filled in by TTP and dCTP with Klenow (exo-; NEB, NE). (B) pRW4′ (12 ng/μl) was incubated in NPE at room temperature. Samples were taken at the indicated times, treated with SDS/proteinase K, and separated on a 1% agarose gel. Lanes 1–4: time points of the reaction in NPE; lane 5: XhoI-digested pRW4 ligated with T4 DNA ligase; lane 6: uncut pRW4; lane 7: pRW4′; lane 8: pRW4′ ligated with T4 DNA ligase. Bands indicated by (*) are NHEJ products. (C) Restriction digestion of the 10-kb repair product (indicated by the line in B). Left: predicted digestion pattern by SalI and EcoRI; middle and right: gel electrophoresis of the digested DNA. The faint bands above the 4.36 band are due to partial digestion. (D) Restriction digestion of the cloned EcoRI fragment. Left: gel electrophoresis of the digested plasmid; right: predicted digestion patterns of the pBR322 plasmid and pRW4 plasmid. X: XhoI site. (E) Gel electrophoresis of the junction DNA directly amplified from the 10-kb repair product. Image published in: Yan H et al. (2005) Copyright © 2005, The Rockefeller University Press. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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