30 Microdissection of Chromosomes and Reverse FISH
389
escence in situ hybridization (FISH), the initial step after collecting the
microdissected fragments is always the amplification by DOP-PCR.
Materials
- Inverted microscope (e.g. Zeiss, Axiovert 135)
Equipment
We recommend the use of an inverted microscope which is equipped
with a long distance condenser. This makes the handling of the dissection needle before and after the dissection procedure as well as the
positioning of the needle much easier. The microscope is equipped
with two objectives: lOx (Plan-Neofluar) for searching for adequate
metaphase spreads and lOOx (Plan-Neofluar, oil-immersion), which
is used to identify chromosomes and to observe and control the actual
microdissection process. In order to align the chromosomes perpendicular to the microdissection needle, a freely rotating working stage is
used.
- Micro-manipulator (Zeiss, Maerzhaeuser, Eppendorf)
We recommend an electronically controlled micro-manipulator for
guiding the microdissection needle.
- Pipette puller (e.g. Narishige PC-lO)
The actual tools for the microdissection are extended solid glass needles. For their preparation solid glass rods (1-2 mm in diameter) are
processed with a standard pipette puller. Preferably a two-step vertical
puller is used. In the first step the glass rod is reduced in diameter and
the second step produces the needles. Usually, both of the needles that
are thus generated can be used.
- Thermocycler
Any thermocycler with or without heated lid can be used. However, as
annealing of the DOP-primer takes place at a relatively low temperature, the cycler must be able to actively chill down the heating block.
- PCR working station
Since the amplification system is initially independent of the priming
sites, any DNA introduced into the reaction is amplified. Hence, maximum care should be taken in order to avoid any DNA contamination.
A PCR working station provides a defined space and is equipped with a
short wave (254 nm) UV-lamp. The UV-lamp can be used to decontaminate the working space itself as well as the tools used such as racks,
tweezers and most importantly, the glass needles.
389
escence in situ hybridization (FISH), the initial step after collecting the
microdissected fragments is always the amplification by DOP-PCR.
Materials
- Inverted microscope (e.g. Zeiss, Axiovert 135)
Equipment
We recommend the use of an inverted microscope which is equipped
with a long distance condenser. This makes the handling of the dissection needle before and after the dissection procedure as well as the
positioning of the needle much easier. The microscope is equipped
with two objectives: lOx (Plan-Neofluar) for searching for adequate
metaphase spreads and lOOx (Plan-Neofluar, oil-immersion), which
is used to identify chromosomes and to observe and control the actual
microdissection process. In order to align the chromosomes perpendicular to the microdissection needle, a freely rotating working stage is
used.
- Micro-manipulator (Zeiss, Maerzhaeuser, Eppendorf)
We recommend an electronically controlled micro-manipulator for
guiding the microdissection needle.
- Pipette puller (e.g. Narishige PC-lO)
The actual tools for the microdissection are extended solid glass needles. For their preparation solid glass rods (1-2 mm in diameter) are
processed with a standard pipette puller. Preferably a two-step vertical
puller is used. In the first step the glass rod is reduced in diameter and
the second step produces the needles. Usually, both of the needles that
are thus generated can be used.
- Thermocycler
Any thermocycler with or without heated lid can be used. However, as
annealing of the DOP-primer takes place at a relatively low temperature, the cycler must be able to actively chill down the heating block.
- PCR working station
Since the amplification system is initially independent of the priming
sites, any DNA introduced into the reaction is amplified. Hence, maximum care should be taken in order to avoid any DNA contamination.
A PCR working station provides a defined space and is equipped with a
short wave (254 nm) UV-lamp. The UV-lamp can be used to decontaminate the working space itself as well as the tools used such as racks,
tweezers and most importantly, the glass needles.