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1. Using a sterile toothpick, inoculate a single isolated bacterial colony into 2 ml TB (or LB) media supplemented with 25 ug/ml kanamycin. Use a 12-15 ml snap-cap polypropylene tube. Grow overnight (up to 16 h) shaking at 225-300 rpm at 37?C.
2. Remove toothpicks using forceps. Centrifuge (SM24 or similar rotor) at 3,000 rpm for
10 min. of spin in the Sorvall. The temperature ot the spin is not critical at this stage.
3. Discard supernatants. Resuspend (vortex) each pellet in 0.3 ml P1 solution. Add 0.3 ml of P2 solution and gently shake tube to mix the contents. Let sit at room temperature for 5 min or so. The appearance of the suspension should change from very turbid to almost translucent.
4. Slowly add 0.3 ml P3 solution to each tube and gently shake during addition. A thick
white precipitate of protein and E. coli DNA will form. After adding P3 solution to every
tube, place the tubes on ice for at least 5 min.
5. Place tubes in the SM24 rotor and spin at 10,000 rpm for 10 min at 4 oC.
6. Remove tubes from centrifuge and place on ice. Transfer supernatant using a P1000
or a disposable pipette to a 1.5 ml eppendorf tube that contains 0.8 ml ice-cold isopropanol. Try to avoid any white precipitate material. Mix by inverting tube a few times; place tubes on ice for at least 5 min. At this stage, samples can be left at -20? C
overnight.
7. Spin in cold microfuge for 15 min.
8. Remove supernatant and add 0.5 ml of 70% EtOH to each tube. Invert tubes several times to wash the DNA pellets. Spin in cold microfuge for 5 min. Optional:repeat step 8.
9. Remove as much of the supernatant as possible. Occasionally, pellets will become dislodged from the tube so it is better to carefully aspirate off the supernatant rather than pour it off.
10. Air dry pellets at room temp. When the DNA pellets turn from while to translucent in appearance, i.e., when most of the ethanol has evaporated, resuspend each in 40 ul TE. Do not use a narrow bore pipets tip to mechanically resuspend DNA sample; rather, allow the solution to sit in the tube with occasional tapping of the bottom of the tube. For large PAC clones resuspension may take over 1 hour.
11. Use 5 ul for digestion with Notl or other rare cutter enzymes. There are Notl sites flanking the Sp6 and T7 promotor regions of the CYPAC2 vector; therefore, this is a very useful enzyme for analysis of insert size and for partial digest restriction mapping. Use 7-10 ul for digestion with a more frequent cutter such as BamHI or EcoRI. |
