The m-PCR amplification of all 3 target gene segments
yielding comparable band intensities was achieved
with the Epicentre FailSafe PreMix Buffer-D and 1 μl
(5 U) of Taq DNA polymerase (Promega). Adjusting the
cycling parameters to include the stepwise reduction
of the annealing temperature from 63 to 54°C considerably
improved the amplification of the 3 target gene
segments and eliminated spurious bands (Fig. 1).
Additionally, resolving the amplified products on 3:1
Nu-Sieve agarose (compared to LE-agarose) and staining
the gel with SYBR-Green-1 nucleic acid stain
(compared to ethidium bromide), improved the sharpness
and intensity of the resolved bands.