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RENATE ULMER
3. Denaturation and hybridization: These two steps are combined in the
procedure described here. Of course a separate denaturation of the
target-sequences gives equal or even better results. Co-denaturation
is time-saving. The adequate denaturation time has to be tested for
each lab.
4. Post-hybridization washes and counterstain: The stringency of the
washing steps has to be evaluated for each lab. In the case where
the signals of probes for chromosomes 13 and 21 are too weak but
signals of probes for chromosomes X,Y, and 18 are good, the stringency
of washing can be reduced for the cosmid probes by using a second
slide for hybridization. The slide with unique DNA-probes for 13 and
21 can be washed with 1xSSC while the a-satellite-probes for X,Y and
18 can be washed more stringently with O.4xSSc.
By using our own DAPI counterstain and mounting medium, we had
better results than with the combined mounting medium ofVysis. The
ready-mixed mounting medium included in the Vysis-kit containing
DAPI should be tested anyway.
5. Signal enumeration: including the points microscopical equipment,
enumeration scan, criteria for interpretation and a warning concerning
maternal cell contamination.
Materials
laboratory
equipment
- incubator (37°C)
- water baths (37±2°C, 74±2°C)
- microcentrifuge
- magnetic stirrer
- microliter pipettor (1-1000 Ill)
- sterile tips (blue and yellow)
- pH meter
- calibrated thermometer
- timer
- forceps
RENATE ULMER
3. Denaturation and hybridization: These two steps are combined in the
procedure described here. Of course a separate denaturation of the
target-sequences gives equal or even better results. Co-denaturation
is time-saving. The adequate denaturation time has to be tested for
each lab.
4. Post-hybridization washes and counterstain: The stringency of the
washing steps has to be evaluated for each lab. In the case where
the signals of probes for chromosomes 13 and 21 are too weak but
signals of probes for chromosomes X,Y, and 18 are good, the stringency
of washing can be reduced for the cosmid probes by using a second
slide for hybridization. The slide with unique DNA-probes for 13 and
21 can be washed with 1xSSC while the a-satellite-probes for X,Y and
18 can be washed more stringently with O.4xSSc.
By using our own DAPI counterstain and mounting medium, we had
better results than with the combined mounting medium ofVysis. The
ready-mixed mounting medium included in the Vysis-kit containing
DAPI should be tested anyway.
5. Signal enumeration: including the points microscopical equipment,
enumeration scan, criteria for interpretation and a warning concerning
maternal cell contamination.
Materials
laboratory
equipment
- incubator (37°C)
- water baths (37±2°C, 74±2°C)
- microcentrifuge
- magnetic stirrer
- microliter pipettor (1-1000 Ill)
- sterile tips (blue and yellow)
- pH meter
- calibrated thermometer
- timer
- forceps