Sample whole-cell diagnostic PCR assays and restriction digests

Copyright information:Taken from "Gene C—Gene Cloning And Tagging for using yeast Homologous Recombination: a novel approach for the analysis of gene expression"Nucleic Acids Research 2005;33(18):e163-e163.Published online 27 Oct 2005PMCID:PMC1270953.© The Author 2005. Published by Oxford University Press. All rights reserved Examples are shown for and . Arrowheads indicate expected PCR products. ( and ) Diagnostic PCR assays to identify correctly cloned target regions in pC1/4. Only clones that are positive for both the left (L) and right (R) junctions are correct (i.e. clones 1 and 3; clone 3). ( and ) Diagnostic PCR assays to identify clones containing the GF-URA3-FP tag (correct clones: clone 3; clone 1). ( and ) Diagnostic PCR assays to identify clones containing a reconstituted GFP tag (correct clones: clones 1–5; clones 2, 4–8). ( and ) Restriction digests of plasmid DNA to confirm the final construct. The restriction enzymes NcoI, XhoI and NdeI cut within the GFP tag and the vector, and tend to cut within the target region (REx represents gene-specific recognition sites), and allow us to differentiate between clones that contain an interrupted GFP tag, a reconstituted GFP tag or no tag.