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JAIME GOSALVEZ et al.
Bal31 Nuclease Bal 31 degrades both the 3' and the 5' termini of duplex DNA,
causing a progressive shortening of double-stranded DNA (dsDNA).
1. Chromosomal DNA is digested overnight according to the standard
subprotocol described for RE digestion.
2. After overnight RE digestion, the slides are washed in 2x SSC (pH 7),
dehydrated in an ethanol series (70%, 85%, 100%) and air dried.
3. The slides are incubated with 5U ofBal31 in 50 III of buffer for 10 min
at 37°C.
4. After Bal 31 digestion, the slides are washed in 2xSSC (pH 7), dehydrated in an ethanol series (70%, 85%, 100%) and air dried.
Notes The action of Bal 31 decreases the amount of double-stranded DNA
(dsDNA), and so, less target DNA will be available for FISH. Thermal denaturation of the slides will be required before "in situ" hybridisation of
the selected probes.
FISH after NU in situ digestion
After extracting and/or modifying the DNA on fIxed chromosomes by the
action of NUs, the DNA remaining on the slide can be "in situ" hybridised
with any DNA probe.
As pointed out above, preparations treated with ExoIII are not thermally denatured, since this enzyme already produces ssDNA for the hybridisation. All other NU-digested preparations require gende denaturation in 50% formamide/2xSSC (pH 7) for 105 s at 67°C. Usually, standard
FISH protocols use higher temperatures and formamide concentrations
for chromosome denaturation. However, since DNA has previously
been "in situ" cleaved, aggressive denaturation would result in massive
DNA loss. Besides this slight modifIcation, the protocols for FISH after
NU-digestion are those currendy used for each material or DNA probe.
See the articles included in this volume for specific FISH procedures.
Results
The present section aims to show a number of classical topics with regard
to genome organisation in eukaryotes where the combined use of "in situ"
digestion with Nucleases and FISH have provided valuable information.
JAIME GOSALVEZ et al.
Bal31 Nuclease Bal 31 degrades both the 3' and the 5' termini of duplex DNA,
causing a progressive shortening of double-stranded DNA (dsDNA).
1. Chromosomal DNA is digested overnight according to the standard
subprotocol described for RE digestion.
2. After overnight RE digestion, the slides are washed in 2x SSC (pH 7),
dehydrated in an ethanol series (70%, 85%, 100%) and air dried.
3. The slides are incubated with 5U ofBal31 in 50 III of buffer for 10 min
at 37°C.
4. After Bal 31 digestion, the slides are washed in 2xSSC (pH 7), dehydrated in an ethanol series (70%, 85%, 100%) and air dried.
Notes The action of Bal 31 decreases the amount of double-stranded DNA
(dsDNA), and so, less target DNA will be available for FISH. Thermal denaturation of the slides will be required before "in situ" hybridisation of
the selected probes.
FISH after NU in situ digestion
After extracting and/or modifying the DNA on fIxed chromosomes by the
action of NUs, the DNA remaining on the slide can be "in situ" hybridised
with any DNA probe.
As pointed out above, preparations treated with ExoIII are not thermally denatured, since this enzyme already produces ssDNA for the hybridisation. All other NU-digested preparations require gende denaturation in 50% formamide/2xSSC (pH 7) for 105 s at 67°C. Usually, standard
FISH protocols use higher temperatures and formamide concentrations
for chromosome denaturation. However, since DNA has previously
been "in situ" cleaved, aggressive denaturation would result in massive
DNA loss. Besides this slight modifIcation, the protocols for FISH after
NU-digestion are those currendy used for each material or DNA probe.
See the articles included in this volume for specific FISH procedures.
Results
The present section aims to show a number of classical topics with regard
to genome organisation in eukaryotes where the combined use of "in situ"
digestion with Nucleases and FISH have provided valuable information.