18 Quantitative DNA Fiber Mapping
239
13. Remove most of the bottom layer and spin again for 3 min.
14. Transfer the top layer to new microcentrifuge tubes and add 400 III
chloroform/isoamyl alcohol (24:1, vol.vol.)
15. Vortex well for 15 s, spin down for 3 min and remove most of the bottom layer followed by a second centrifugation for 3 min.
16. Transfer top layer to a new microcentrifuge tube, add 2.5 volumes, i.e.,
1 ml 100% ethanol and let the DNA precipitate for 30 min at -20°e.
17. Spin down for 15 min, discard the supernatant and wash the pellet in
ice cold 70% ethanol, spin again briefly, remove supernatant and air
dry the pellet.
18. Resuspend the pellet in 20-40 III TE, pH 7.4 containing 10 Ilg/ml RNase.
19. Incubate 30 min at 37°C [in water bath]; then, store at -20°C until used.
2. Preparation of DNA from Yeast Artificial Chromosome (YAe) clones
Retrieve the desired yeast clone containing the YAC from the library and
grow it on AHC agar for 2-3 days at 30°e. Pick colonies from the plate and
culture the clones in up to 35 ml AHC media at 30°C for 2-3 days.
1. Centrifuge cells (in -35 ml AHC media) at 2000g at 4°C for 5 min.
2. Decant the supernatant and resuspend cells in 3 ml total of 0.9 M sorbitol, O.IM EDTA, pH 7.5, containing 4111 ~-mercaptoethanol, followed
by addition of 100 III of zymolase (2.5 mg/ml), and then incubate at
37°C for 60 min.
3. Pellet the cells at 2000 g and 4°C for 5 min and decant supernatant.
4. Resuspend pellet in 5 ml of 50 mM Tris, pH 7.4,20 mM EDTA. Add
0.5 ml of 10% SDS and mix gently. Incubate at 65°C for 30 min.
5. Add 1.5 ml of 5 M potassium acetate and place on ice for 60 min.
6. Spin at 12,000 gfor 15 min at 4°C, and transfer the supernatant to a new
tube.
7. Mix the supernatant gently with 2 volumes of 100% ethanol by inverting the tube a few times. Spin at 5000 rpm (2000g) for 15 min at room
temperature.
DNA extraction,
phenol purification
and alcohol
precipitation
239
13. Remove most of the bottom layer and spin again for 3 min.
14. Transfer the top layer to new microcentrifuge tubes and add 400 III
chloroform/isoamyl alcohol (24:1, vol.vol.)
15. Vortex well for 15 s, spin down for 3 min and remove most of the bottom layer followed by a second centrifugation for 3 min.
16. Transfer top layer to a new microcentrifuge tube, add 2.5 volumes, i.e.,
1 ml 100% ethanol and let the DNA precipitate for 30 min at -20°e.
17. Spin down for 15 min, discard the supernatant and wash the pellet in
ice cold 70% ethanol, spin again briefly, remove supernatant and air
dry the pellet.
18. Resuspend the pellet in 20-40 III TE, pH 7.4 containing 10 Ilg/ml RNase.
19. Incubate 30 min at 37°C [in water bath]; then, store at -20°C until used.
2. Preparation of DNA from Yeast Artificial Chromosome (YAe) clones
Retrieve the desired yeast clone containing the YAC from the library and
grow it on AHC agar for 2-3 days at 30°e. Pick colonies from the plate and
culture the clones in up to 35 ml AHC media at 30°C for 2-3 days.
1. Centrifuge cells (in -35 ml AHC media) at 2000g at 4°C for 5 min.
2. Decant the supernatant and resuspend cells in 3 ml total of 0.9 M sorbitol, O.IM EDTA, pH 7.5, containing 4111 ~-mercaptoethanol, followed
by addition of 100 III of zymolase (2.5 mg/ml), and then incubate at
37°C for 60 min.
3. Pellet the cells at 2000 g and 4°C for 5 min and decant supernatant.
4. Resuspend pellet in 5 ml of 50 mM Tris, pH 7.4,20 mM EDTA. Add
0.5 ml of 10% SDS and mix gently. Incubate at 65°C for 30 min.
5. Add 1.5 ml of 5 M potassium acetate and place on ice for 60 min.
6. Spin at 12,000 gfor 15 min at 4°C, and transfer the supernatant to a new
tube.
7. Mix the supernatant gently with 2 volumes of 100% ethanol by inverting the tube a few times. Spin at 5000 rpm (2000g) for 15 min at room
temperature.
DNA extraction,
phenol purification
and alcohol
precipitation