15 Cloning Vectors
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Procerlure
1. Preparation of dialysis tubing: Heat the dialysis tubing ( cut in pieces of ln advance
approximately 25 cm) in 2% Na2HC03, 1 mM EDTA (pH 8.0) and boil
for 1 min. After cooling rinse the tubing first in distilled water then in
ethanol. Rinse the dialysis tubing in 1 mM EDTA (pH 8.0) and store it in
this solution at 4°C.
2. Inoculate 100 ml ofLB medium with a single colony ofJM 103 (grown on Day 1
a minimal agar plate). Grow the cells with shaking at 37°C to an OD 600 of
1.0, then store these cells at 4°C.
3. Inoculate 100 ml of LB medium with a M13 phage plaque. Grow with
shaking at 37°C overnight.
4. To 900 ml of2 x YT medium add 100 ml ofJM103 culture from step 2. Day 2
Grow the cells with shaking to an OD600 of 1.5.
5. Add 100 ml of the phage stock from step 3 and shake 15 min at 37°C.
6. Add 0.5 ml of chloramphenicol solution. Shake 1-2 hours at 3rC (accumulation of RF DNA).
Preparation of closed circular M13 RF DNA (Triton-lysozyme method)
7. Centrifuge the cells at 4000 x g for 10 min at 4°C.
8. Discard the supernatant and resuspend the cells in 30 ml of solution A.
9. Add 1 ml of a freshly made lysozyme solution (30 mg/ml).
10. Incubate at room temperature for 30 min.
11. Add 3 ml of solution B.
12. Incubate at room temperature for 30 min.
13. Centrifuge at 27,000 x g for 20 min at 4°C.
14. Pour the supernatant in a graduated cylinder. Add 1 g CsCl for each ml
of the supernatant and dissolve the CsCl.
15. In Quick-seal ultracentrifuge tubes, add ethidium bromide solution (11
20 volume of the DNA/CsCl solution) and the DNA/CsCl solution from
step 14. If there is a remaining volume, fill with CsCl solution to the
constriction. Balance the tubes and dry the inlets. Seal the tubes
with the prewarmed sealer and allow the sealing caps to cool completely