ISOLATION OF PLASMID (PROBE) DNA FOR IN SITU HYBRIDIZATION (ISH)
- Inoculate a 2-mL culture in a Kaput tube from a single colony and
place it on a 37°C shaker overnight.
- Inoculate a 400-mL culture in a 1 L flask by pouring the 2-mL preculture
into the flask; allow 4 to 5 hrs incubation at 37°C while shaking.
- Harvest the cells by centrifugating for 10 min at 6,000 rpm at 4°C.
Decant the supernatant and place at an angle on ice to allow the remaining
liquid to drain away from the pellet. Remove the remaining liquid
with a sterile pipette after several minutes.
- Resuspend the pellet in 5 mL of solution I (50 mM glucose, 25 mM
Tris pH 8.0, 10 mM EDTA); and transfer the culture into a 50-mL Nalgene
polyallomer centrifuge tube.
- Add 10 mL of solution II (O.2 N NaOH, 1 % SDS), cover tube with
Parafilm, and gently invert repeatedly to mix; allow the solution
to set on ice for 10 min.
- Add 7.5 mL of solution III (3 M NaAc pH 4.8), cover tube with parafilm,
and mix by inverting gently.
- Centrifuge tube at 12,000 rpm for 15 min at 4°C, pour the supernatant
into a second 50-mL tube, add 11-mL isopropanol, cover with parafilm
and mix by inverting gently centrifuge tube for 5 min at 5,000 rpm
at 4°C, decant supernatant, rinse the pellet in 70% ethanol; allow
the tube to drain for 10 min, lypholize to near dryness; disslove
the pellet in 4.5-mL TE.
- Add 4.7 g of CcCl and 0.4 mL of a 10 mg/ml ethidium bromide solution
in TE (the density of the sample is between 1.55 and 1.59 g/mL, add
TE or CsCl to adjust; plasmid DNA without CsCl purification also can
be used for biotin-labeling, however better results are obtained when
CsCl purified plasmid DNA is used).
- Tranfer the solution into a Beckman '13 x51' polyallomer quick-seal
tube, fill to just below the neck (the tube now should weigh 9.45-9.6
g), and seal.
- Centrifuge tube at 20°C at 65,000 rpm for 4 hrs or 55,000 rpm
- Remove the plasmid band from tube with a needle and syringe, and
transfer to a 1.5-mL tube.
- Add 1 volume of saturated isopropanol (mix equal volumes of isopropanol
and 20 X SSC pH 7.0) and mix well. Centrifuge, or allow to stand for
several minutes. Remove upper phase with pipette.
- Repeat the extraction 4 to5 times, or until the pink color in the
- Dilute the DNA sample with 2 volumes of TE, add 1/10 volume of 3M
NaAc pH 7 and mix well.
- Add 2 volumes of 95% ethanol and mix well.
- Centrifuge for 5 min, drain tube, rinse the pellet and sides of
the tube with 70% ethanol, drain the ethanol, and dry the pellet.
- Dissolve the DNA into 400 µL TE, add 20 µL RNase A (10
mg/mL), mix, and incubate for 15 min at room temperature.
- Add an equal volume of a 1:1 phenol:chloroform solution, mix well,
centrifuge briefly, and transfer the upper phase to another tube.
- Add an equal volume of chloroform, mix well, centrifuge briefly,
and transfer the upper phase to another tube.
- Precipitate the DNA with 3 M NaAc and ethanol and dissolve the pellet
in 400 µL TE.
- Determine the DNA concentration by using spectrophotometer or by
electrophoresis on a minigel.