To identify targeted ES cell clones, we developed a robust LR-PCR system that uses one set of reaction conditions for every targeted allele screened. In addition, we used an in-house primer generation program (“Primer Brain”) to generate genome-specific primers for the LR-PCR. Primers were selected from 2-kb blocks of sequence upstream of the 5′ homology arm (GF) and downstream of the 3′ homology arm (GR) and from a variable-sized region that contains the critical exon (EX). Primers were first extracted by a single-base-pair tiling of each region into 24- to 30-mers that end in G/C, have at least 10 G/C bases and have a melting temperature of at least 64 °C. Primer choice was weighted negatively to avoid both ‘runs’ of nucleotides (for example, ‘AAA’) and self-annealing ends. The top 100 high-scoring primers in each region were aligned against the current mouse genome (NCBIM37) with Exonerate software (http://www.ebi.ac.uk/~guy/exonerate) and were weighted negatively based on the number of alignments to the genome, with added negative weight given to alignments close to the 3′ end of primers. The two best-scoring primers from each block (GF1 and GF2; GR1 and GR2; EX1 and EX2) were grouped and primer combinations (for example, GF1 and EX1) were screened to eliminate pairs with a 4-bp overlap at their 3′ ends. The resulting GF, GR and EX primers were stored in an Oracle database.