DNA Isolation from P1 Artificial Chromosome (PAC) Clones
(Adapted for Pieter de Jong's laboratory, 8/93)

This is a rapid alkaline lysis miniprep method for isolating DNA from large PAC clones. It is a modification of a standard Qiagen Tip method that uses no organic extractions or columns. The method works very well for doing analytical restriction digests of PAC clones and can be scaled up if necessary.

I. Solutions

II. Method

1. Inoculate a single isolated bacterial colony into 2ml TB (or LB) media supplemented with 25µg kanamycin. Use a 12-15 ml snap-cap polypropylene tube. Grow overnight (up to 16h) shaking at 225-300 rpm at 37^oC.

2. Centrifuge (SM24 or similar rotor) at 3,000 rpm for 10 min in a Sorvall centrifuge (or equivalent). The temperature of the spin is not critical at this stage.

3. Discard supernatants. Resuspend (vortex) each pellet in 0.3ml P1 solution. Add 0.3ml of P2 solution and gently shake tube to mix the contents. Leave at room temperature for 5 min or so. The appearance of the suspension should change from very turbid to almost translucent.

4. Slowly add 0.3ml 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 to a 1.5ml Eppendorf tube that contains 0.8ml ice-cold isopropanol. Try to avoid any white precipitated 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^oC overnight.

7. Spin in cold microfuge for 15 min.

8. Remove supernatant and add 0.5ml 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 tube so it is better to aspirate off the supernatant carefully rather than pour it off.

10. Air dry pellets at room temp. When the DNA pellets turn from white to translucent in appearance, i.e. when most of the ethanol has evaporated, resuspend each in 40 µl TE. Do not use a narrow bore pipette tip to resuspend DNA sample; rather, allow the solution to sit in the tube with occasional tapping of the bottom of the tube.

11. Use 5 µl for digestion with NotI or other rare cutter enzymes. There are NotI 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 µl for digestion with a more frequent cutter such as BamHI or EcoRI