() The targeting mini-library
Hiroshi Sakamoto, Sabine Thiberge, Akerman Susan, Janse, Chris J., Carvalho, Teresa Gil, Robert Ménard · Figshare · 2011
Copyright information:Taken from "Towards systematic identification of essential genes by transposon shuttle mutagenesis"Nucleic Acids Research 2005;33(20):e174-e174.Published online 10 Nov 2005PMCID:PMC1283541.© The Author 2005. Published by Oxford University Press. All rights reserved The mini-library consists of 20 replacement plasmids, which share identical homology arms and the selectable marker, but differ by the length of a central tag (from 0.3 to 1.2 kb). The sizes of the homology arms after plasmid restriction digestion with EcoRI or HincII + PmlI are shown above. E, EcoRI; H, HincII; N, NheI; F, FseI; P, PmlI. () Frequencies of DCO events at the genomic locus. Parasites were electroporated with the mini-library, using various amounts and lengths of homology of the targeting DNA and the Gene Pulser II (Biorad) or the Nucleofector™ (Amaxa) electroporators. () PCR analysis of a transfected parasite population. Parasites were transfected with 50 µg of the mini-library having long homology arms using a Gene Pulser II (Biorad) electroporator. PCR analysis uses a forward primer hybridizing to the 5′ end of the tag and a reverse primer hybridizing to the 5′ end of the right homology arm (A). The PCR on the left uses the pool of transfected DNA (Input DNA) as a template, and the PCR on the right uses the genomic DNA of the resistant parental population (Par. Pop.) as a template. () Southern blot analysis of the locus in the wild-type and parasites transfected with the mini-library. Genomic DNA was cut with ClaI, which does not cut in the central region of the constructs, and hybridized with a probe corresponding to the 5′ part of the coding sequence. The size of the ClaI fragment in the WT (2.1 kb) increases in the parental population (DCOs) by the size of the central region of the constructs (1.9–2.8 kb).