30
Chromosome, Microdissection and Microcloning
Video
Zeiss IM35
X/Y scanner
Monitor
Laser-microbeam apparatus
Hamamatsu
image pro-
cessor [1966]
TV-camera
Dye laser
FL 2002
Lambda Physik
Excimer laser
EMG 103 MSC
Lambda Physik
Photo-camera
Autofocus
control board
X/Y scanner
control board
Drive control
Fig. 3
Diagram of the laser-microbeam microdissection apparatus.
The microinstruments necessary for fragment collection and
manipulation (not depicted) are essentially the same as
described earlier.
3.3
Dissection and Collection of Chromosome
Fragments by the Videomicroscopy Method
Approximately two days before microdis-
section, cultures are initiated to prepare
high-mitotic index cells according to stan-
dard procedures. Metaphase spreads are
prepared a few hours before microdissec-
tion and stored in ethanol. Chromosomes
are GTG band–stained immediately be-
fore the scheduled microdissection. Volu-
metric micropipettes (i.d.
'
1–2
µ
m) and
straight microdissection needles (o.d.
=
0
.
2–0.5
µ
m) are prepared at precalibrated
settings on a micropipette puller, Flled
with parafFn oil, and mounted on the ap-
propriate holders. ±or chromosomal DNA
collection, 200
µ
L of Flter-sterilized paraf-
Fn or silicone oil is placed on a depression
slide or moist chamber near the dissection
slide. Similarly, nanoliter drops of pro-
teinase K/SDS and Tris-HCl solutions are
placed under oil using volumetric pipettes.
Also, several 1 to 2 mL drops of aqueous
Tris-HCl/EDTA buffer are placed adjacent
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