Procedure:

1. Take 30-50µL of tear sample (collected directly from eye) in a fresh microfuge tube.
2. Add 250μL Buffer GDTE1. Incubate the tube at 850C for 15 min and vortex after every 2 min.
3. Add 250μL Buffer GDTE2 and mix by inverting the tube 4–6 times. Place the tube at 850C again for another 15 minutes.
4. Add 350μL Buffer GDTE3 and invert the tube immediately. Shake vigorously to mix the solutions, DO NOT VORTEX AT THIS STAGE. Vortex may cause shearing of genomic DNA.
5. Centrifuge for 10 min at 13,000 rpm (~10000xg) in a table-top micro centrifuge. A compact pellet will form.
6. Collect the supernatant in a fresh microfuge tube and add 1/5th volume of isopropanol. Incubate at room temperature for 2 minutes.
7. Apply the isopropanol added solutions to the GMini Spin Column by decanting or pipetting. Avoid mixing of cell debris with the supernatant as this may clog GMini spin Column thus lowering the DNA yield.
8. Centrifuge at 13,000 rpm (~10000xg) for 30–60 s. Discard the flow-through.
9. Wash GMini Spin Column by adding 600μL Wash Buffer* and centrifuging for 30–60 s as previously.
10. Discard the flow-through.
11. Repeat step 10. If required, repeat wash step once again.
12. Discard the flow-through, and centrifuge for an additional 2 min to remove residual wash buffer from membrane.
    This step is extremely important to ensure complete removal of ethanol. Presence of ethanol in purified DNA may inhibit subsequent enzymatic reactions.
13. Place the GMini Spin Column in a clean 1.5mL micro centrifuge tube (not provided). To elute DNA, add 50μL nuclease free water to the center of each GMini Spin Column, let stand for 1 min, and centrifuge for 1 min at maximum speed (~10000Xg) on a table top micro centrifuge.
14. Discard the column and collect the eluted DNA present in micro centrifuge tube.

.