1. To ensure reproducibility of DAF patterns, the same thermocycler and reagents should be used. The interpretation of weak bands can be problematic, and typing should be duplicated in separate runs to help overcome this problem (see Subheading 1.).
2. A minority of Campylobacter isolates may be nontypable by DAF or PFGE owing to the production of DNAses. This can be overcome by routinely treating bacterial suspensions with formaldehyde before proceeding with the typing method (see Subheading 2.1.).
3. Caution: Formaldehyde is toxic and harmful if inhaled or absorbed through skin or mucosa. This reagent should only be handled in a safety cabinet with extraction ventilation (see Subheading 3.1.).
Fig. 5. PFGE profiles from three Campylobacter spp. SmaI macrorestricted fragments ranging from 24 to 267 kbp are resolved by this method.
4. Microscopic examination by dilute carbol fuchsin staining. Prepared by dissolving 20 g of carbol fuchsin (BDH Chemicals, Poole, England) and liquefying 100 g of phenol (BDH Chemicals) together in a 3-L conical flask. Add 200 mL of absolute ethanol and 1715 mL of distilled water. Filter the resultant solution and dilute in sterile distilled water to give a 10% working carbol fuchsin solution (see Subheadings 3.1. and 3.3.1.).
5. Round-bottomed microcentrifuge tubes facilitate emulsification of organisms (see Subheading 3.1.)
6. Caution: care should be taken when handling phenol-chloroform. As phenol is an acid, it can cause skin burns. This reagent should only be handled in a safety cabinet with extraction ventilation (see Subheading 3.1.).
7. Ensure that none of the white layer at the interface of the phenol chloroform and DNA solution is disturbed (see Subheading 3.1.).
8. DNA suspensions may be stored at 4°C for up to 3 mo, or, alternatively, may be aliquoted and frozen at -20°C and thawed once prior to use (see Subheading 3.1.).
9. Optimal temperature for SmaI is 25-30°C (see Subheading 3.3.3.).
10. Up to four gel slices may be cut for digestion in 100 ^L total volume of restriction solution (see Subheading 3.3.3.).
11. Alternatively, agarose slices may be allowed to digest overnight (see Subheading 3.3.3.).
12. Prepare sufficient gel volumes according to the PFGE apparatus used. Quoted volume applies to the Gene Navigator System (Pharmacia Biotech, Uppsala, Sweden), having an actual gel size of 152 mm2 (see Subheading 3.3.4.).
13. Use blotting paper to blot dry the surface of wells, facilitating the loading of plug slices into the gel (see Subheading 3.3.4.).
14. Unused agarose can be allowed to solidify and can be reheated several times as needed (see Subheading 3.3.4.).
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