Relative quantification to a reference gene (GAPDH) was performed using the ΔΔCt method (see Supplementary material for primer pairs).
Typically, about 3 kb of genomic DNA sequence immediately upstream of the predicted ATG initiator site, was used as the putative promoter (see supplementary material for primer sequences).
To detect the CAG-Cre-ERTM construct, PCR of genomic DNA was carried out (see Supplementary material for primers).
This was cloned into the replication-defective C. difficile vector pMTL82151 (20), suitable for pseudosuicide in C. difficile (4, 5), using standard cloning techniques, resulting in the plasmid pAFP1200 (see Table S2 in the supplemental material for primers and restriction enzymes).
Inverse PCR Life Technologies Platinum Taq DNA Polymerase High Fidelityy; see supplementary material S1, Supplementary Material online, for primers) using pairs of outward-facing primers positioned at the ends of contig_578, contig_74, contig_85, and contig_131 resulted in positive amplification products that, when sequenced, closed the contigs upon themselves.
First, primers were designed to amplify from S. pombe genomic DNA approximately 300 bp of 5′ and 3′ sequences flanking each open reading frame (ORF) targeted for deletion (see Materials and Methods for primer design and Table S1, for primer sequences).
Maternal haplotypes of 19 bulls were determined by sequencing the entire D-loop (supplementary table S2, Supplementary Material online, for primers), and sequences were aligned using ClustalW (Larkin et al. 2007).
See supplementary material Table S6 for primer sequences.
Expression of genes (see supplementary material Table S1 for primer sequences) was analysed by quantitative PCR.
Mutations in fragment 4 were introduced through PCR primers (see supplementary material Table S6 for primer sequences).
