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

282 Malorny and Helmuth
Table 2
Volumes for Amplification Controls
Components Vol/reaction
Vol for 22X
master mixture
(stock concentration) (final concentration) (for 20 reactions)
PCR-grade water 8.6 µL 189.2 µL
10X PCR buffer 2.5 µL 55 µL
dNTP Mixture (2 mM) 2.5 µL (200 µM) 55 µL
Primer HB10 (10 pmol/µL) 1.0 µL (0.4 µM) 22 µL
Primer HB11 (10 pmol/µL) 1.0 µL (0.4 µM) 22 µL
Magnesium chloride (50 mM) 0.75 µL (1.5 mM) 16.5 µL
Fraction V BSA (10 mg/mL) 2.5 µL (1 µg/µL) 55 µL
pUC19 DNA (1 fg/µL) 1.0 µL (1 fg) 22 µL
Taq DNA polymerase (5 U/µL) 0.15 µL (0.75 U) 3.3 µL
DNA extraction template 5.0 µL
Total vol 25.0 µL
3.4. Southern Blot
In order to check if the Salmonella-specific PCR product has been formed,
Southern blotting, with subsequent hybridization, should be conducted as
described in the following protocols.
PCR products separated by gel electrophoresis are transferred onto a positively
charged nylon membrane and hybridized against a DIG-labeled PCR probe. The
transfer of PCR products onto the membrane can be performed by capillary,
vacuum, or electroblotting techniques, all of which are standard laboratory protocols
(23–24). Here, we describe a Southern transfer using a vacuum blotter.
1. After documentation, rinse the agarose gel with distilled water and incubate it in
500 mL of 0.25 M HCl for 15 min at room temperature with gentle agitation.
2. Pour off the HCl and rinse the gel with distilled water. Add 500 mL of 0.5 N
NaOH and shake as in step 1 for 30 min.
3. Pour off the NaOH solution and place the gel in 2X SSC buffer.
4. Cut a piece of the nylon membrane large enough to cover the gel surface.
5. Set up the vacuum blotter according to the manufacturer’s instructions. Generally,
a set-up includes a Whatman 3MM paper of appropriate size, the nylon
membrane, a window plastic gasket, the agarose gel, and the sealing frame. Overlay
the arrangement with 10X SSC buffer. Apply vacuum no more than 5 mm Hg
for 45 min.
6. After the transfer, rinse the nylon membrane in 2X SSC buffer (see Note 6). Dry
the membrane between Whatman 3MM paper and place it DNA-side-down on a