Fig. 2. An agarose gel (1.5% w/v) showing the DNA profiles of a collection of unrelated Campylobacter spp. isolates.
3. The reaction conditions used for amplification are listed in Table 2.
4. Integron PCR products were analyzed by conventional agarose gel (1.5% w/v) electrophoresis (Fig. 4).
A one-day standardized laboratory protocol for the molecular subtyping of Campylobacter spp. by PFGE (adapted for Campylobacter spp. from the PulseNet Method The National Molecular Subtyping Network for Foodborne Disease Surveillance for nontyphoidal Salmonella) was applied in this study.
3.3.1 Preparation of Agarose Plugs for Pulsed-Field Gel Electrophoresis
1. Cultures of Campylobacter spp. to be investigated should be well grown (48-72 h) on Preston medium and checked microscopically for purity (see Note 4 and Fig. 1).
2. Emulsify the plate contents of each isolate in a 1-mL volume of 0.85% (w/v) NaCl in sterile 2-mL microcentrifuge tubes. Centrifuge at 11,000g for 5 min.
3. Decant the supernatant and resuspend isolates in 0.9 mL 0.85% (w/v) NaCl.
4. Add 100 ^L of formaldehyde (37-40% v/v), mix, and leave at room temperature for 1 h.
5. Centrifuge for 5 min at 11,000g and then decant the supernatant. Wash the pellet three times in 1 mL 0.85% (w/v) NaCl.
6. Resuspend the pellet in 2 mL of cell suspension buffer.
7. Adjust the volume of the cell suspension buffer to give an optical density of 1.8-2.0 at 610 nm.
8. Prepare agarose plugs as follows: weigh 0.25 g SKG into a 50-mL screw-capped tube (Sarstedt). Add 23.5 mL TE buffer and mix gently. Dissolve the agarose completely by microwaving.
9. Place tubes in a 50°C water bath for 5 min and then add 1.25 mL of 20% (w/v) SDS (preheated to 50°C). Mix well by gentle inversion of the capped tube.
10. For each isolate, transfer 0.4 mL of the pure bacterial culture suspension (at room temperature) to a 1.5-mL Eppendorf tube, add 20 ^L of Proteinase K (20 mg/mL) to each tube, and mix gently by pipeting.
11. Add 0.4 mL of the SKG/SDS mixture to each bacterial culture and again mix by pipeting gently up and down several times. Place in a 50°C waterbath.
12. Aspirate or pipet each sample into the barrel of a prelabeled syringe. Cover the nozzle of each syringe with parafilm and place on ice for 15 min to allow the plugs to solidify.
3.3.2. Cell Lysis
1. Label 17 x 120-mm sterile tubes with appropriate isolate numbers.
2. Add 5 mL of the cell lysis buffer to each tube, containing 25 ^L of the stock Proteinase K. Calculate the total volume of the cell lysis/Proteinase K buffer required and aliquot 5 mL of this mixture into the prelabeled tubes.
3. Trim the agarose plugs with a sterile scalpel and add the whole plug/or plug cut in two segments to the lysis buffer.
4. Incubate in a 54°C waterbath for 1.5-2 h, with constant shaking at 175-200 rpm. Ensure that the water level in the bath is above the level of the lysis buffer in the tubes.
5. Heat sufficient sterile distilled H2O to 50°C to allow plugs to be washed twice with 1015 mL of water.
6. Remove tubes from waterbath and carefully pour off lysis buffer into an appropriate discard container.
7. Add 10-15 mL of preheated sterile distilled H2O to each tube and shake the tubes vigorously in a 50°C waterbath for 10-15 min.
8. Again pour off the water from the plugs and repeat this wash step with preheated water, ensuring that no lysis buffer remains on the rims or caps of tubes.
9. Pour off water, add 10-15 mL preheated sterile TE buffer, and shake the tubes vigorously in a 50°C waterbath for 10-15 min.
10. Pour off TE and repeat wash step with preheated TE three more times.
11. Decant last wash and add 5 mL sterile TE. At this stage it is possible to continue with the restriction digest, or plugs can be stored at 4°C until ready to proceed.
3.3.3. Restriction Digest of DNA in Agarose Plugs With Sma/
1. Label the appropriate number of 1.5-mL microcentrifuge tubes.
2. Prepare the SmaI digest reactions (see Note 9) according to the table below and add the mixtures into the microcentrifuge tubes:
Sterile distilled H2O 87.5
10X digest buffer J 10
Total volume 100
3. Cut 1.5-2-mm wide slices from each plug with a sterile scalpel blade and transfer to the tubes containing the digest mixture (see Note 10).
4. Mix by tapping gently and ensure that plug slices are fully submerged in the mixture.
5. Incubate samples in 25-30°C waterbath for a minimum of 2 h (see Note 11).
3.3.4. Preparation of Agarose Gels and Sample Loading
2. Prepare 3 L of 0.5X TBE buffer by mixing 150 mL of the 10X TBE stock buffer with 2850 mL distilled water.
3. Add 1.1 g SKG agarose to 110 mL of the 0.5X TBE buffer in a 250-mL flask and mix gently (see Note 12).
4. Cover loosely with parafilm and microwave for 60 s; mix gently and repeat for 15-s intervals until the agarose is completely dissolved. Cool to approx 60°C and pour into a precasting gel apparatus. Allow to solidify for approx 30 min and then gently remove the comb.
5. Place the gel into the electrophoresis tank and overlay completely with 0.5X TBE buffer. Pre-electrophorese the gel for 1 h.
6. After the pre-electrophoresis step, remove the gel from the electrophoresis tank.
7. Remove digested plug slices from 37°C water bath. Gently decant the enzyme/buffer solution, taking care not to score the plug slices with the pipet tip, and add 200 ^L 0.5X TBE to stop the digestion reaction. Incubate at room temperature for 5 min.
8. Remove one plug slice from each tube at a time and load into the preformed wells in the agarose gel (see Note 13).
9. Prepare 20 mL of a mixture containing 1% (w/v) SKG agarose in 0.5X TBE as follows: Add 0.2 g agarose to 20 mL 0.5X TBE and microwave until dissolved. Seal the gel wells by overlaying with this molten agarose solution (see Note 14).
10. Replace the loaded gel back into the gel tank, submerging it in 0.5X TBE electrophoresis buffer, and apply an electric current of 200 V to perform the electrophoresis. DNA fragments are separated using a ramped pulse switching every 0.5-25 s for 20 h (23), at 10.5°C (Fig. 5).
3.3.5. Staining and Documentation of PFGE Agarose Gel
Ethidium bromide solution is at a final concentration of 0.5 ^g/mL in distilled water. Stain the gel for 20-30 min in a covered container. Destaining may be performed if required in distilled water.
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