3.3. Qualitative analysis of DNA
Present protocol (Method 4) overcomes the issue of difficulty in lysing
the thick oocyst wall and further removing the contaminating agents that
act as PCR inhibitors. The four critical changes incorporated with this
protocol; 2.5% CTAB, freeze at -80ºC and boil at 95ºC as
‘snap-chill-boil’ instead of ‘freeze-thaw’ and proteinase-K steps,
followed by treatment with DNase-Free-RNase mix to enhance the DNA
quality and final precipitation of DNA with isopropanol and also
considerably reducing the time taken for the total process. The results
showed that the modified DNA extraction protocol in the current study
generated DNA with high purity and workability, as evidenced by the
absence of RNA contamination during gel electrophoresis (Figure 2A) and
Nested PCR amplification of 18SSU rRNA gene showed theCryptosporidium positive faecal on 1.8% agarose gel stained with
ethidium bromide respectively (Figure 2B).
(Figure 2A and 2B here)