1. Prepare a 2% agarose gel (w/v) by adding 1.75 g of agarose to 70 mL deionized water, plus 1.4 mL 50X TAE buffer and 3.5 ^L ethidium bromide (10 mg/mL).
2. Once the gel is set, place it in the tank with the electrophoresis buffer just covering the surface of the gel.
3. Add 4 ^L of the loading dye into each sample, gently resuspend, and load 12 ^L of the samples into the well. Also load the molecular weight markers.
4. When loading is complete, place the lid on the tank and plug into the power pack.
5. Run the gel at a constant voltage of 100-120 V until the dye front approaches the edge of the gel.
6. Switch off the power pack and remove the gel tray.
7. Put on the UV eye shield.
8. Place the gel on the UV transilluminator and switch on.
9. Visualize and photograph the products (see Note 7).
1. Each section of the methods should be performed either in separate laboratories or in different areas to avoid contamination. Preparation of the master mixes should be carried out in a specimen-free laboratory. Inoculation of the first- and second-round mix tubes should be performed in separate laminar flow cabinets to prevent cross-contamination. Analysis of the PCR products should also be performed in a separate area or laboratory.
2. Gloves should be worn at all times to avoid contamination with DNAses and RNAses.
3. When preparing the master mixes, overestimating the final required volume by one or two reactions to allow for volume losses and pipeting errors is recommended. Also, make a minimum batch of 25 reaction mixtures for the first and second rounds. Large batch numbers are recommended, as very small volumes of the primers are used.
4. It is recommended that the following be used as positive and negative controls. E. rhusiopathiae ATCC 19414 can be used as the positive control for both PCR methods, and E. tonsillarum ATCC 43339 can be used as the negative control for the E. rhusiopathiae -specific PCR.
5. A DEPC water negative control should be used for both PCR methods.
6. Make sure the lids on the centrifuge and microcentrifuge tubes are placed on firmly to prevent evaporation of the suspension during boiling or PCR amplification.
7. Samples should be tested twice on separate days to check for reproducibility. If discrepant results are obtained, then the analysis should be repeated. If the third PCR is positive, then the overall results are considered positive. If the third PCR is negative, then the overall result is considered negative.
This work was supported by the Western Australian Fisheries Industry Council and a grant from the Fisheries Industry Research and Development Corporation.
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