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)