Info

PFGE, pulsed-field gel electrophoresis.

(a) and (b), different electrophoretical conditions evaluated for Salmonella serotypes Enteritidis and Typhimurium.

PFGE, pulsed-field gel electrophoresis.

(a) and (b), different electrophoretical conditions evaluated for Salmonella serotypes Enteritidis and Typhimurium.

3.3 Infrequent-Restriction-Site PCR

1. Grow the isolates in 20 mL of trypticasein soy broth at 37°C overnight with shaking.

2. Spin at 3000g for 20 min. Resuspend the pellet in 10 mL TE buffer to wash the cells and spin again.

3. Resuspend the pellet with 500 ^L of lysis solution and transfer to an Eppendorf tube.

4. Add 250 ^L ammonium acetate, mix, and leave on ice for 10 min.

5. Add 500 ^L of chloroform/2-pentanol and shake by hand vigorously for 10 min.

6. Spin at 11,600g for 10 min and transfer 740 ^L of supernatant to a fresh Eppendorf tube. Add 400 ^L of cold isopropanol, mix by inverting the tube for 1 min, and spin at 11,600g for 5 min.

7. Wash the DNA three times with 1 mL of ice-cold 70% (v/v) ethanol and vacuum dry.

8. Rehydrate the DNA in 100 ^L of TE buffer and keep at 4°C overnight.

9. Double-restrict 1 ^g of bacterial DNA with 10 U of Xbal and 10 U of Hhal endonucleases (or the same amount of TaqI), with the appropriate buffer in a 12.5-^L final volume for 1 h at 37°C. If Taql restriction endonuclease is used, carry out a second restriction step for 1 h at 65°C.

10. Add the following reagents to the reaction: 2 U T4 DNA ligase and corresponding buffer, l mM ATP, 20 pM of each adapter (see Note 7) in a final reaction volume of 20 ^L. Carry out the DNA ligation at 16°C for 1 h.

Table 4

PCR Fingerprinting Amplification Temperatures

Table 4

PCR Fingerprinting Amplification Temperatures

Temperature (°C)

Time (min)

No. of cycles

ERIC2 primer

Was this article helpful?

0 0

Post a comment