6 In Situ Hybridization for DNA: Fluorescent Probe
73
- Master Mix (of Hybridization)
- 5.5 ml formamide
- 19 dextran sulfate
- 0.5 ml 20X SSC
- These are heated to 70°C for several hours to dissolve the
dextran sulfate, cooled, pH adjusted to 7.0 and the volume
brought to 7 ml. This volume is 70% of that used in the final
hybridization mix. The remaining 30% of the volume are
filled with probe(s), carrier DNA, and waterif needed. Used
in this way Master Mix gives a hybridization mixture that
is 55% formamidellO% dextran sulfatellXSSC.
Procedure
1. Bring Coplin jar containing the denaturation solution (70%
formamide/2XSSC) to room temperature. Place the jar in a
74°C water bath 30 mins prior to use in order to attain the
solution temperature. Measure the temperatures of the solution inside the Coplin jar, and make sure the temperature is
:snoc.
2. Thaw reagents at room temperature, then centrifuge each
tube for a few secs using a bench-top micro centrifuge.
3. Prepare ethanol series in three Coplin jars: 50ml1 00% ethanol
into one jar, 50ml 85% ethanol into another, and 50ml 70%
ethanol into the last.
FISH using fluorochrome-directly-labeled DNA probes
Note: Perform all steps, which do not require light for manipulation (incubation periods, washes, etc.), in darkness in order to
avoid photo bleaching of fluorochrome-directly-labeled probes.
Slides containing cells are prepared using methanol-acetic acid Remarks
(3:1) fixation.
Heat or enzymatic treatment may induce loss of adherence of
cells and tissue sections to glass slides. This may be overcome by
using aminoalkylsilane-coated glass slides.
73
- Master Mix (of Hybridization)
- 5.5 ml formamide
- 19 dextran sulfate
- 0.5 ml 20X SSC
- These are heated to 70°C for several hours to dissolve the
dextran sulfate, cooled, pH adjusted to 7.0 and the volume
brought to 7 ml. This volume is 70% of that used in the final
hybridization mix. The remaining 30% of the volume are
filled with probe(s), carrier DNA, and waterif needed. Used
in this way Master Mix gives a hybridization mixture that
is 55% formamidellO% dextran sulfatellXSSC.
Procedure
1. Bring Coplin jar containing the denaturation solution (70%
formamide/2XSSC) to room temperature. Place the jar in a
74°C water bath 30 mins prior to use in order to attain the
solution temperature. Measure the temperatures of the solution inside the Coplin jar, and make sure the temperature is
:snoc.
2. Thaw reagents at room temperature, then centrifuge each
tube for a few secs using a bench-top micro centrifuge.
3. Prepare ethanol series in three Coplin jars: 50ml1 00% ethanol
into one jar, 50ml 85% ethanol into another, and 50ml 70%
ethanol into the last.
FISH using fluorochrome-directly-labeled DNA probes
Note: Perform all steps, which do not require light for manipulation (incubation periods, washes, etc.), in darkness in order to
avoid photo bleaching of fluorochrome-directly-labeled probes.
Slides containing cells are prepared using methanol-acetic acid Remarks
(3:1) fixation.
Heat or enzymatic treatment may induce loss of adherence of
cells and tissue sections to glass slides. This may be overcome by
using aminoalkylsilane-coated glass slides.