238
HEINZ-ULRICH G. WEIER
Procedure
1. Alkaline lysis protocol and purification of DNA from Pl, PAC or BAC clones
This protocol describes the isolation of DNA from -20 ml overnight cultures using 40 ml Oakridge centrifugation tubes. The protocol can be
scaled down to accommodate smaller volumes.
1. Grow culture overnight in -30 ml LB or TB medium containing the
recommended amount of antibiotic.
2. Prepare Oakridge tubes. Write the clone ID on a small piece of tape
stuck to the cap. Spin 18.5 ml of culture at 2000 g for 10 min at
4°e and discard the supernatant.
3. Resuspend the pellet in 2340 !J.I of AL Solution I, then add 100 !J.I of
lysozyme stock to each tube. Incubate tubes for 5 min at room temperature. Then, place the tubes on ice.
4. Add 5.2 ml of AL Solution II. The mixture should now become clear.
Mix gently by inverting the tubes several times. Incubate for 5 min on
ice.
5. Add 3.8 ml of AL Solution III and mix gently by inverting the tubes
several times. Incubate for 10 min on ice.
6. Spin for 15 min at high speed (11,500 rpm/14,000g).
7. Transfer 10.4 ml of supernatant into a new Oakridge tube, add 5.8 ml of
isopropanol and mix gently by inverting the tubes several times. Use
the old cap [with the ID stickerI on the new tube.
8. Spin for 5 min at -10,000 g and discard the supernatant. Watch the
pellet!
9. Wash the pellet in cold 70% ethanol. Let the pellets dry briefly, i.e., for
-20-40 min at 20
0 e to 37°C.
10. Resuspend the pellet in 0.8 ml of TE buffer and split the volume into
two 1.5 ml microcentrifuge tubes.
11. Add 400 !J.I phenol/chloroform/isoamyl alcohol to each tube. All centrifugations during the following phenol/chloroform extraction are
done at 12,000 g.
12. Vortex for 15 s and spin down for 3 min.
HEINZ-ULRICH G. WEIER
Procedure
1. Alkaline lysis protocol and purification of DNA from Pl, PAC or BAC clones
This protocol describes the isolation of DNA from -20 ml overnight cultures using 40 ml Oakridge centrifugation tubes. The protocol can be
scaled down to accommodate smaller volumes.
1. Grow culture overnight in -30 ml LB or TB medium containing the
recommended amount of antibiotic.
2. Prepare Oakridge tubes. Write the clone ID on a small piece of tape
stuck to the cap. Spin 18.5 ml of culture at 2000 g for 10 min at
4°e and discard the supernatant.
3. Resuspend the pellet in 2340 !J.I of AL Solution I, then add 100 !J.I of
lysozyme stock to each tube. Incubate tubes for 5 min at room temperature. Then, place the tubes on ice.
4. Add 5.2 ml of AL Solution II. The mixture should now become clear.
Mix gently by inverting the tubes several times. Incubate for 5 min on
ice.
5. Add 3.8 ml of AL Solution III and mix gently by inverting the tubes
several times. Incubate for 10 min on ice.
6. Spin for 15 min at high speed (11,500 rpm/14,000g).
7. Transfer 10.4 ml of supernatant into a new Oakridge tube, add 5.8 ml of
isopropanol and mix gently by inverting the tubes several times. Use
the old cap [with the ID stickerI on the new tube.
8. Spin for 5 min at -10,000 g and discard the supernatant. Watch the
pellet!
9. Wash the pellet in cold 70% ethanol. Let the pellets dry briefly, i.e., for
-20-40 min at 20
0 e to 37°C.
10. Resuspend the pellet in 0.8 ml of TE buffer and split the volume into
two 1.5 ml microcentrifuge tubes.
11. Add 400 !J.I phenol/chloroform/isoamyl alcohol to each tube. All centrifugations during the following phenol/chloroform extraction are
done at 12,000 g.
12. Vortex for 15 s and spin down for 3 min.