Isolation From BAC & PAC Clones
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.
(filter sterilized, 4oC)
50mM Tris, pH 8
10 mM EDTA
100 ug/ml RNase
sterilized, room temp)
3M KOAc, pH 5.5
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
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
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.