3.1. Modified DNA extraction procedure (Method 4)
The final DNA extraction protocol was developed which gives a higher yield that also facilitates better purity of the DNA. Briefly, described the procedure for efficient DNA extraction, a total of 500 µl of faecal lavage/washing (Suspended in 1X PBS) was taken in a 2 ml microcentrifuge tube. The microcentrifuge tube was further added with 800 µl of CTAB isolation buffer (100mMTris-HCl, pH-8; 1.5M NaCl, 25mM EDTA (no salt); 2.5% CTAB) and vortexed for 30 seconds. The mixture was frozen in a -80ºC deep freezer for 15minutes. The frozen mixture was transferred to a -20C cooler to carry and immediately given heat shock in a water bath at 95ºC for 5 minutes. This cycle was repeated two more times (a total of 3 cycles of freeze and boil) with brief vortexing for 30 seconds between each cycle. The mixture was centrifuged at 12000rpm for 5 minutes at room temperature (RT; 28°C). Following this, ~800 µl supernatant was carefully collected and transferred to a new sterile 1.5 ml microcentrifuge tube and spiked with 10 µl of DNase-Free-RNase mix (Cat# 12091021, Invitrogen). The mixture was then incubated in the water bath at 37°C for 20 minutes followed by the addition of an equal volume (~800 µl) of Phenol: chloroform: isoamyl alcohol (25:24:1) mixture and mixed vigorously through vortexed for 30 seconds till the content turned white/milky emulsion and centrifuged (conditions as above). Approximately ~600µl of the upper aqueous phase was collected in a new microcentrifuge tube and added an equal volume of chloroform: isoamyl alcohol (24:1) reagent mixed vigorously through vortexed to form a white emulsion and centrifuged again. 500µl of the upper aqueous phase was transferred in a new microcentrifuge tube and added 0.8 volume of (~400 µl) of isopropanol. The content of the tube was mixed by gently inverting the tubes several times and incubating the mixture at -20°C for 30 minutes, centrifuging at 12000rpm for 10 minutes at room temperature. The supernatant was carefully discarded without disturbing the DNA pellet at the bottom. The pellet was further washed with 1 ml of 70% ice-cold ethanol (70% ethanol + 30% Nuclease free water V/V) and centrifuged; the step was repeated. After washing, ethanol was removed by carefully decanting the content and the pellet was dried in the fume hood for 5 minutes. The DNA pellet was re-suspended in 30-40µl of TE buffer (pH 8) and stored at -20ºC for further molecular analysis.