Chromosome, Microdissection and Microcloning
27
3
25
70
Oil chamber
Prot K - SDS
+
Restriction enzyme
+
Vector
+
Ligase
In vitro
packaging
µ
L volumes
+
Phenol
+
CHCI
3
Microdissection
35
6
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
e
Fig. 1
Oil chamber microdissection apparatus: (a) dimensions of the oil chamber
(mm) and (b)–(h) steps of microcloning operations.
3.2.2
Videomicroscope Method
We have described a chromosome work-
station that can be used for microdissec-
tion and
in situ
hybridization experiments
(Fig. 2a). The station features an inverted
microscope equipped with a ±xed stage, a
low-level light video camera, and a video
monitor. A 12 V, 100 W halogen lamp
and a high-focal length phase-contrast
condenser were added to enhance illu-
mination and obtain a better resolution
of chromosomal GTG bands. In addition,
a new microscope stage was designed,
featuring a mechanical stage with
XY
coordinates graduation and a circular ro-
tating stage in the center (Fig. 2b). The
circular stage accommodates the holders
for the dissection and collection slides.
For easy relocation of a speci±c chro-
mosomal spread, the center stage is also
graduated 360
. The circular stage offers
the advantage of rapidly positioning tar-
get chromosomes perpendicular to the
dissecting needle tip regardless of their
orientation in the spread on the slide.
Also, the stage accepts large coverslips
(35 mm
×
50 mm), thus maximizing the
numberofspreadsonaslideaswellasthe
number of chromosome fragments col-
lected per session.
Video cameras have an inherent magni-
±cation factor that may range from 10
×
to 200
×
. For example, the video cam-
era series QX-104 (VSP Inc.) and the
charge-coupled device (CCD) video cam-
era CCD-72 (Dage Corp.) have an intrinsic
magni±cation factor of 40
×
.Whencou
-
pled with a microscope using a 40 or 63
×
objective, we were able to achieve magni±-
cation of metaphase chromosomes on the
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