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

294 Wastling and Mattsson
Fig. 1. B1-nested PCR products separated on an ethidium bromide-stained agarose
gel and visualized under UV illumination. MW, molecular weight marker; lane 1,
positive control; lane 2, negative control; lane 3, peripheral blood sample taken from a
sheep 10 d after experimental infection with T. gondii tachyzoites.
2. Load all samples, including controls, on a 2.0% agarose gel containing 0.5 µg/mL
ethidium bromide and 1X TAE buffer. Separate the samples at 5 V/cm for about
40 min.
3. Analyze and record the gel image under UV illumination.
3.3.2. Product Verification through Southern Blotting
1. Transfer DNA from the agarose gel in 3 M NaCl and 8 mM NaOH onto a nylon
membrane by capillary blotting.
2. After the transfer mark the positions of each sample well and the positions of the
molecular weight standards.
3. Wash the membrane with 2X SSC.
4. Cross-link DNA to the wet membrane according to the manufacturer’s instructions.
The membrane can be used immediately or can be stored dry at 4°C for
future use.
5. To prepare the probe: assemble a PCR with primers P1 and P4 using the PCR
DIG Probe synthesis kit according to the manufacturer’s instructions. Use 100 ng
T. gondii genomic DNA as template.
6. Separate the labeled PCR product on a 1% agarose gel. Use a clean scalpel to
excise the gel segment containing the PCR product.
7. Purify the DNA from the agarose gel using GENECLEAN according to the
manufacturer’s instructions.
8. Prehybridize the membrane in DIG Easy Hyb solution for 2 h. Use 20 mL
prehybridization solution per 100 cm 2 membrane.