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ANNETTE DEICHMANN AND KLAUS DEICHMANN
before removing. Check the tubes for leakage by pressing gently in the
middle ofthe tube. Place them in the rotor (e.g. VTi50 from Beckman).
Note: Be careful when handling ethidium bromide because it is a very carcinogenic agent. Furthermore, UV light in the presence of ethidium bromide causes nicks in the closed circular DNA. Therefore take care of light
conditions and expose the gradients as briefly as possible to the UV light.
16. Centrifuge at 225,000 x g for 16 hrs at 20°C (e.g. VTi50 at 48,000 rpm).
Day 3 17. Stop the centrifuge without brake.
18. Remove a tube carefully and place it in a clip. Using a UV -lamp you will
see 2 bands. The upper band contains nicked circular and linear M13
DNA and linear fragments of the bacterial chromosome. The lower
band contains the desired closed circular M13 RF DNA.
19. Pierce the top of the tube with a 20-gauge needle to allow air to enter.
With a second 20-gauge needle pierce the tube just below the lower
band and immediately collect the solution dripping out of the needle
which contains the closed circular DNA. After the lower band is collected, discard the rest of the gradient.
20. Place the collected material in smaller Quick-seal tubes and add 1/20
volume of ethidium bromide solution. Fill with CsCl solution. Seal the
tubes and place them in the rotor (e.g. VTi65 from Beckman).
21. Centrifuge at 278,000 x g for at least 8 hrs at 20°C (e.g. VTi65 at 54,000
rpm).
Day 4 22. Stop the centrifuge without brake.
23. Remove a tube carefully and visualize the DNA under UV light. You
should see one thick band which contains the desired phage DNA
and only a small upper band or no upper band. Collect the DNA as
described in step 19.
24. To the collected material add an equal volume of CsCl- saturated isopropanol (use only the isopropanollayer). Vortex and discard the isopropanollayer which is now colored pink and contains the ethidium
bromide. Repeat this step four more times until the isopropanol remains clear.
25. Dialyze the DNA against TE buffer for 4 hours: Remove a piece of dialysis tubing from the stock (see step 1) and clip one end. Insert the DNA
solution and clip the other end ofthe dialysis bag. Place the bag in 1-21
ANNETTE DEICHMANN AND KLAUS DEICHMANN
before removing. Check the tubes for leakage by pressing gently in the
middle ofthe tube. Place them in the rotor (e.g. VTi50 from Beckman).
Note: Be careful when handling ethidium bromide because it is a very carcinogenic agent. Furthermore, UV light in the presence of ethidium bromide causes nicks in the closed circular DNA. Therefore take care of light
conditions and expose the gradients as briefly as possible to the UV light.
16. Centrifuge at 225,000 x g for 16 hrs at 20°C (e.g. VTi50 at 48,000 rpm).
Day 3 17. Stop the centrifuge without brake.
18. Remove a tube carefully and place it in a clip. Using a UV -lamp you will
see 2 bands. The upper band contains nicked circular and linear M13
DNA and linear fragments of the bacterial chromosome. The lower
band contains the desired closed circular M13 RF DNA.
19. Pierce the top of the tube with a 20-gauge needle to allow air to enter.
With a second 20-gauge needle pierce the tube just below the lower
band and immediately collect the solution dripping out of the needle
which contains the closed circular DNA. After the lower band is collected, discard the rest of the gradient.
20. Place the collected material in smaller Quick-seal tubes and add 1/20
volume of ethidium bromide solution. Fill with CsCl solution. Seal the
tubes and place them in the rotor (e.g. VTi65 from Beckman).
21. Centrifuge at 278,000 x g for at least 8 hrs at 20°C (e.g. VTi65 at 54,000
rpm).
Day 4 22. Stop the centrifuge without brake.
23. Remove a tube carefully and visualize the DNA under UV light. You
should see one thick band which contains the desired phage DNA
and only a small upper band or no upper band. Collect the DNA as
described in step 19.
24. To the collected material add an equal volume of CsCl- saturated isopropanol (use only the isopropanollayer). Vortex and discard the isopropanollayer which is now colored pink and contains the ethidium
bromide. Repeat this step four more times until the isopropanol remains clear.
25. Dialyze the DNA against TE buffer for 4 hours: Remove a piece of dialysis tubing from the stock (see step 1) and clip one end. Insert the DNA
solution and clip the other end ofthe dialysis bag. Place the bag in 1-21