7. Centrifuge at 15,000 g for 10 min.
8. Remove the upper layer and to which add chloroform: isoamylalcohol in the ratio of 24:1, mix and centrifuge at 15,000 g for
10 min.
9. Separate the upper phase.
10. Add double volume of ethanol and 3 M ammonium precipitate
for better DNA precipitation.
11. Freeze the mixture for 2 h and centrifuge at 15,000 g for
30 min.
12. Dry the DNA precipitated from aqueous phase and resuspend
in TE buffer.
13. Pellet the DNA precipitate in a microcentrifuge and dry at
room temperature in vacuum.
14. Resuspend the acquired pellet in 50 μl of TE-buffer.
15. Store the isolated extracted DNA at 4
C until further use.
16. Run the DNA sample by agarose gel electrophoresis along with
1 kb markers of lambda DNA as standard.
17. Load DNA sample of 5 μl on 0.8% agarose gel at 100 mA for
30 min and the gel is observed under UV light.
5.2 Agarose
Preparation for Gel
Casting
1. Add agarose (0.5 g) to 50 ml of 1Â TAE buffer. This represents
1% agarose gel.
2. Boil agarose to dissolve absolutely until clear solution appear
and cool to lukewarm (60
C) and add 0.5 μg/ml ethidium
bromide.
3. Place the comb of electrophoresis set, approximately 2 cm away
from the cathode.
4. Pour the agarose without creating any bubbles, cool it for
20 min. The thickness of the gel should be around 0.5–0.9 cm.
5. Add 1Â TAE buffer into the gel tank until the buffer level
stands at 0.5–0.8 cm over the surface of gel and slowly take off
the comb.
6. Connect the power cord to the electrophoretic power pack
setup. Run the gel for 1 h at 100 V.
7. Load 9 μl of DNA sample mixed with the loading dye (1 μl),
10 μl control DNA and 10 μl marker into the well using a
pipette.
8. Switch off the current when the bromophenol blue (the tracking dye) has progressed 1 cm above the bottom end and
disconnect the power supply.
9. Gently push the gel onto the UV transilluminator in gel
documentation.
88
Laboratory Techniques for Entomopathogenic Nematodes
8. Remove the upper layer and to which add chloroform: isoamylalcohol in the ratio of 24:1, mix and centrifuge at 15,000 g for
10 min.
9. Separate the upper phase.
10. Add double volume of ethanol and 3 M ammonium precipitate
for better DNA precipitation.
11. Freeze the mixture for 2 h and centrifuge at 15,000 g for
30 min.
12. Dry the DNA precipitated from aqueous phase and resuspend
in TE buffer.
13. Pellet the DNA precipitate in a microcentrifuge and dry at
room temperature in vacuum.
14. Resuspend the acquired pellet in 50 μl of TE-buffer.
15. Store the isolated extracted DNA at 4
C until further use.
16. Run the DNA sample by agarose gel electrophoresis along with
1 kb markers of lambda DNA as standard.
17. Load DNA sample of 5 μl on 0.8% agarose gel at 100 mA for
30 min and the gel is observed under UV light.
5.2 Agarose
Preparation for Gel
Casting
1. Add agarose (0.5 g) to 50 ml of 1Â TAE buffer. This represents
1% agarose gel.
2. Boil agarose to dissolve absolutely until clear solution appear
and cool to lukewarm (60
C) and add 0.5 μg/ml ethidium
bromide.
3. Place the comb of electrophoresis set, approximately 2 cm away
from the cathode.
4. Pour the agarose without creating any bubbles, cool it for
20 min. The thickness of the gel should be around 0.5–0.9 cm.
5. Add 1Â TAE buffer into the gel tank until the buffer level
stands at 0.5–0.8 cm over the surface of gel and slowly take off
the comb.
6. Connect the power cord to the electrophoretic power pack
setup. Run the gel for 1 h at 100 V.
7. Load 9 μl of DNA sample mixed with the loading dye (1 μl),
10 μl control DNA and 10 μl marker into the well using a
pipette.
8. Switch off the current when the bromophenol blue (the tracking dye) has progressed 1 cm above the bottom end and
disconnect the power supply.
9. Gently push the gel onto the UV transilluminator in gel
documentation.
88
Laboratory Techniques for Entomopathogenic Nematodes