PCR Detection of Microbial Pathogens PCR Detection of Microbial ...

170 Gallien
10. Ethidium bromide: 1% (w/v) stock solution (Roth, Karlsruhe, Germany). Dilute
1:1000 with distilled water to a final concentration of 10 mg/L. Alternatively,
Gelstar staining solution (Biozym) can be used. Dilute the stock solution 1:100 in
distilled water.
11. DNA molecular weight markers II, V, and IX (Roche Diagnostics, Mannheim,
Germany) containing the following fragments (in bp): Marker II : 125; 564; 2027;
2322; 4361; 6557; 9416; 23130; and Marker V: 8; 11; 18; 21; 51; 57; 64; 80; 89;
104; 123; 124; 184; 192; 213; 234; 267; 434; 458; 504; 540; 587; and Marker IX:
72; 118; 194; 234; 271; 281; 310; 603; 872; 1078; 1353.
12. Gel loading buffer: 62.5 mg bromophenol blue, 62.5 mg xylene cyanol, 6.25 g
Ficoll ® (Type 400; Sigma), make up to 25 mL with distilled water.
2.3. DNA Hybridization and Digoxigenin Labeling of Probes
2.3.1. Preparation of Agar Plates and Nylon Membranes
1. Solution for denaturation: 0.5 M sodium hydroxide, 1.5 M sodium chloride.
2. Solution for neutralization, pH 7.4: 1 M Tris-HCl, 1.5 M sodium chloride.
3. Solution for equilibration (20X SSC): 3 M sodium chloride, 0.3 M sodium citrate,
pH 7.0.
4. Proteinase K: 1 mg/mL, solution in 2X SSC.
2.3.2. Digoxigenin Labeling of Probes
1. Water: autoclaved, free of DNase.
2. Primers: MK1/MK2, KS7/KS8, LP43/LP44; SK1/ SK2, working solution 50
pmol/µL (see Table 1).
3. dNTP mixture: see Subheading 2.2., step 4
4. Digoxigenin-11-2'-deoxy-uridine-5'-triphosphate (DIG-11-dUTP): 1 mM.
5. DNA polymerase, PCR buffer, MgCl 2: see Subheading 2.2.
6. Agarose: low-melting grade (Serva, Heidelberg, Germany).
7. TAE buffer (50X): see Subheading 2.2., item 8.
2.3.3. Prehybridization and Hybridization
1. DIG Easy Hyb ® solution (Roche Diagnostics) (see Note 2).
2. DIG-labeled probes for stx 1, stx 2, and eae (at least 25 ng/mL for 100 mm 2 of
membrane disc).
3. Reference strains: E. coli C 600 (stx and eae negative), E. coli C 600 J1 (stx 1
positive), E. coli C 600 W 34 (stx 2 positive), E. coli 161 - 84 (stx 1 , stx 2, and
eae positive).
2.3.4. Washings and Detection
1. Washing solution 1: sodium dodecyl sulfate (SDS) 1 g/L, 2X SSC (see Subheading
2.3.1., item 3).
2. Washing solution 2: SDS 1g/L, 0.5X SSC.
3. DIG Nucleic Acid Detection Kit (Roche Diagnostics).
4. Buffer 1 (maleic acid buffer): 0.1 M maleic acid (11.6 g/L), 0.15 M NaCl (8.77 g/L).