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

180 Gallien
Fig. 3. Photographic image showing a master plate in the upper part and a replica
nylon membrane after hybridization in the lower part (see Subheadings 3.3.1. and
3.3.5.).
the plate with 2 mL of physiological saline and prepare appropriate dilutions in
water. Use these suspensions as template in a screening PCR again.
2. It is possible to use another solution (called solution B) for prehybridization and
hybridization. It contains the following components: 5 X SSC solution (see Subheading
2.3.1.), 0.1 % (w/v) N-lauroylsarcosine, 0.1% (w/v) SDS, Blocking
reagent ® (Roche Diagnostics). To prepare the solution, dissolve N-lauroylsarcosine
and SDS in 5X SSC. Calculate and add the amount of blocking reagent. Heat to
70°C and stir the solution for 1 h. The solution remains opaque. When applying
solution (‘B’), only use the following solution for all washing steps: 0.1% (w/v)
SDS in 0.04X SSC. It is possible to use the solution containing labeled probes 2 or
3 irrespectively of the kind of hybridization solution (DIG Easy Hyb or B). For
reuse, heat the solution to 70°C and chill on ice.