246
ANNETTE DEICHMANN AND KLAUS DEICHMANN
ln advance
Standard reaction
• Incubator
• Vortex
Procedure
1. Pick a single colony from the agar plate using a sterile toothpick. Let the
toothpick drop in 2 ml of LB medium containing the appropriate antibiotic. Repeat this for as many colanies as you wish to work up. Grow
the cells with shaking overnight at 37°C.
2. The following day prepare solution B and add the lysozyme to solution
A.
3. Fill a 1.5 ml microcentrifuge tube with the overnight culture from step 1.
Store the remainder of the culture at 4 oc and use later to make a frozen
bacterial stock of the positive clones.
4. Centrifuge the cells in the Eppendorf centrifuge for 20 sec.
5. Resuspend the pellet in 100 Jll of solution A. Incubate for 5 min at room
temperature.
6. Add 200 Jll of solution B. Place on ice for 5 min.
7. Add 150 Jll of solution C. Mix carefully and place on ice for 5 min.
8. Centrifuge for 2 min.
9. Save the supernatant in a new microcentrifuge tube and discard the
pellet.
10. Add 150 Jll ofphenol and 150 Jll of chloroform/isoamyl alcohol (24/1).
Vortex and centrifuge for 10 sec.
11. Pipet the supernatant containing the plasmids into a new microcentrifuge tube. Add 1/10 volume of solution C and 2 volumes of95% ethanol.
Mix well and incubate at room temperature for at least 2 min.
12. Centrifuge in the Eppendorf centrifuge for 2 min.
13. Discard the supernatant carefully and wash the pellet in 1 ml of 70%
ethanol.
14. Centrifuge for 2 min.
15. Discard the ethanol and air dry the pellet for 5 min.
ANNETTE DEICHMANN AND KLAUS DEICHMANN
ln advance
Standard reaction
• Incubator
• Vortex
Procedure
1. Pick a single colony from the agar plate using a sterile toothpick. Let the
toothpick drop in 2 ml of LB medium containing the appropriate antibiotic. Repeat this for as many colanies as you wish to work up. Grow
the cells with shaking overnight at 37°C.
2. The following day prepare solution B and add the lysozyme to solution
A.
3. Fill a 1.5 ml microcentrifuge tube with the overnight culture from step 1.
Store the remainder of the culture at 4 oc and use later to make a frozen
bacterial stock of the positive clones.
4. Centrifuge the cells in the Eppendorf centrifuge for 20 sec.
5. Resuspend the pellet in 100 Jll of solution A. Incubate for 5 min at room
temperature.
6. Add 200 Jll of solution B. Place on ice for 5 min.
7. Add 150 Jll of solution C. Mix carefully and place on ice for 5 min.
8. Centrifuge for 2 min.
9. Save the supernatant in a new microcentrifuge tube and discard the
pellet.
10. Add 150 Jll ofphenol and 150 Jll of chloroform/isoamyl alcohol (24/1).
Vortex and centrifuge for 10 sec.
11. Pipet the supernatant containing the plasmids into a new microcentrifuge tube. Add 1/10 volume of solution C and 2 volumes of95% ethanol.
Mix well and incubate at room temperature for at least 2 min.
12. Centrifuge in the Eppendorf centrifuge for 2 min.
13. Discard the supernatant carefully and wash the pellet in 1 ml of 70%
ethanol.
14. Centrifuge for 2 min.
15. Discard the ethanol and air dry the pellet for 5 min.