Croning et al. BMC Genomics 2010, 11:74 http://www.biomedcentral.com/1471-2164/11/74 Page 6 of 7 Figure 4 Experimental validation of Southern blot probes. Each lane on the four blots A-D contains mouse genomic DNA extracted from a separate embryonic stem (ES) cell line, that was restriction-digested and size-separated on an 0.6% by gel electrophoresis before blotting. The gel was run for 16-18 hr at 20-22 V, and a molecular weight standard of HindIII-digested Lambda DNA was employed. Panels A-D were hybridized with probes designed against four distinct mouse loci situated near the Iqsec1, Igsf9, Iqsec3 and Gda genes in order to confirm whether homologous recombination had taken place, ablating or modifying the gene’s function. In each case examples of wild-type (WT) and gene-targeted (TGT) DNA are revealed by the presence of the extra band in the TGT ES clones, at a differing molecular weight. order to confirm homologous recombination had occurred thus correctly targeting the gene to be ablated as part our of our high-throughput mouse knockout and molecular neurobiological phenotyping programme [14]. 13/16 of the probes tested gave a usable signal upon blotting, the remainder gave a smear likely indicative of non-specific probe binding, or no resolvable signal. Representative Sothern blots are shown in Figure 4. Conclusions We have developed an automated system for the effec- tive design of Southern blot probes. Many candidate probes that lie in a given genomic window are searched against the target genome in a brute-force approach to finding the best probe in the locus, as assessed by uniqueness and repetitive DNA sequence content. Using these in silico measures we can automatically design probes that would be predicted to perform as well, or better, than previous manual designs, while reducing the time taken by the molecular biologist to yield a success- ful probe. The majority of the probes we tested experi- mentally in Southern blotting performed well confirming our in silico prediction methodology, and the usefulness of the software for automated genomic Southern blot probe design. Availability and requirements Project name: southern_blot Project home page: http://www.genes2cognition. org/software/southern_blot and Additional file 1. Operating system(s) UNIX and Linux variants Programming language: Perl and SQL Other requirements: BioPerl core 1.5.0 or higher, BioPerl run 1.4 or higher, Ensembl core 32 or higher, Config::IniFiles 2.38 or higher, DBI 1.32 or higher, GD 2.17 or higher, Exonerate 1.0.0, Primer3 1.0.0, LSF 5.1 or higher, MySQL 5.045 or higher License: Artistic License 2.0 Any restrictions to use by non-academics: none Additional file 1: Software package for automated design of genomic Southern blot probes. Archive of all the components of the pipeline packaged using “tar”, and subsequently compressed with “gzip”. Includes source code, example configuration files, example output, and a user’s guide for installation. Click here for file