DNA Isolation from BAC, PAC & YAC Clones
This is a rapid alkaline lysis miniprep method for isolating DNA
from large construct harbouring 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 and can be scaled up if necessary.
I. Solutions
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P1 (filter sterilized, 4°C)
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P2 (filter sterilized, room
temp.)
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15 mM Tris, pH 8
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0.2N NaOH
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10 mM EDTA, pH 8
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1% SDS
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100 µg/ml RNAse A
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P3 (autoclaved, 4°C)
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3M KOAc, pH 5.5
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II. Method
- Inoculate a single isolated bacterial colony into 2ml TB (or
LB) media supplemented with relevant antibiotic. Use a 12-15 ml
snap-cap polypropylene tube. Grow overnight (up to 16h) shaking at
225-300 rpm at 37°C.
- 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.
- 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.
- 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.
- Place tubes in the SM24 rotor and spin at 10,000 rpm for 10 min
at 4°C.
- 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°C overnight.
- Spin in cold microfuge for 15 min.
- 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.
- 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.
- 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.