3.2.3. DNA Amplification Fingerprinting (DAF) Analysis
Each DNA template was analyzed in duplicate by DAF to ensure the reproducibility of the DNA profile and to provide reliable data quality.
1. Each PCR assay incorporated the 10-mer oligonucleotide primer HLWL85 for DAF analysis.
2. For each reaction combine 5.0 ^L 25 mMMgCl2, 5.0 ^L 10X PCR buffer, 8 ^L working stock dNTPs to give a final concentration of 0.2 mM dNTP, 200 ng genomic DNA, and 2.5 U Taq DNA polymerase.
3. The primer HLWL85 (Table 1) was used at a concentration of 50 pmol per reaction in a final reaction volume of 50 ^L.
4. The reaction conditions used for amplification are listed in Table 2. Expected product sizes are variable but may range from 180 to 6000 bp (see Fig. 2 and ref. 22).
3.2.4. Molecular Detection of Ciprorofloxacin Resistance in Campylobacter Isolates
1. Amplifications were performed in 50-^L reaction volumes containing 20 pmol of C. jejuni and 10 pmol of C. coli the correct primer pair (Table 1), 200 ^M each dNTP, and 1.5 mM MgCl2.
2. A 2.5-^L volume of 10X Taq DNA polymerase buffer and 2.5 U Taq DNA polymerase were added.
3. Approximately 33 ng of DNA template, corresponding to 1 ^L were added to the reaction mixture.
4. The reaction conditions used for amplification are listed in Table 2.
5. Expected product sizes: C. coli: 192-bp product, (ACT^ATT) mutation (Fig. 3A); C. jejuni: 265-bp product, (ACA^ATA) mutation (Fig. 3B).
3.2.5. Integron PCR Analysis
1. The integron primer sequences (10) used to identify the presence of integron structures are listed in Table 1.
2. In a final reaction volume of 50 ^L, the following was added; 5 ^L 10X PCR buffer, 5 ^L 25 mM MgCl2, 8 ^L dNTP working stock solution, 25 pmol of each IntF and IntR primer (Table 1), 200 ng template DNA, and 2.5 U Taq DNA polymerase.
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