204
BURT G. FEUERSTEIN et al.
4. Run 3-5 ~l of probe on a 1% agarose gel. Fresh DNA size should run
from 300-2300 bp. Formalin-fixed DNA should run from approximately 200-600 bp.
5. Store labeled probes at -20°C.
ISubprot ocol 2
Metaphase production
The art of human metaphase preparation is outside the scope of this chapter. We refer the reader to Chapter 3 of this manual for details. However, a
few general comments concerning human metaphases for CGH should be
made.
After human lymphocyte cultures have been prepared, a cabinet that
controls the temperature and the humidity of the environment and allows
more reproducible spreads to be produced is used (Thermatron CDS-5
Cytogenetic Drying Chamber, Holland, MI) (8). We use a temperature
of 25°C and vary the humidity from 40-55% depending on the quality
of metaphase spreads. This can be monitored directly with a phase microscope, starting with the humidity at 55% and decreasing it stepwise until
spreads are category II or category III (9, 10). From experience, the optimal humidity is near 47.5%. Since DAPI bands identify chromosomes for
analysis, chromosome structure must be preserved in order to utilize them
for CGH. The slides are aged for several days before storing in a freezer
under nitrogen; the preparations can be used for more than six months.
If the experimenter does not wish to prepare his/her own human metaphases, fairly good spreads prepared specifically for CGH experiments
can be purchased (Vysis, Inc. Downers Grove, IL). However, since methods for preparing mouse metaphases are not generally available, we provide a method here that we use for CGH.
Materials
Reagents - Complete medium: Sterile fIlter 450 ml Dulbecco's modified Eagle
medium (F-12/H-21 0:1),50 ml fetal calf serum, 7.5 ml 1M HEPES,
5 ml 0.2 M glutamine, 5 ml pen/strep (10,000 U/ml penicillin and 10,000
mg/ml strepomycin).
- Hank's Balanced Salt Solution, Calcium and Magnesium Free
BURT G. FEUERSTEIN et al.
4. Run 3-5 ~l of probe on a 1% agarose gel. Fresh DNA size should run
from 300-2300 bp. Formalin-fixed DNA should run from approximately 200-600 bp.
5. Store labeled probes at -20°C.
ISubprot ocol 2
Metaphase production
The art of human metaphase preparation is outside the scope of this chapter. We refer the reader to Chapter 3 of this manual for details. However, a
few general comments concerning human metaphases for CGH should be
made.
After human lymphocyte cultures have been prepared, a cabinet that
controls the temperature and the humidity of the environment and allows
more reproducible spreads to be produced is used (Thermatron CDS-5
Cytogenetic Drying Chamber, Holland, MI) (8). We use a temperature
of 25°C and vary the humidity from 40-55% depending on the quality
of metaphase spreads. This can be monitored directly with a phase microscope, starting with the humidity at 55% and decreasing it stepwise until
spreads are category II or category III (9, 10). From experience, the optimal humidity is near 47.5%. Since DAPI bands identify chromosomes for
analysis, chromosome structure must be preserved in order to utilize them
for CGH. The slides are aged for several days before storing in a freezer
under nitrogen; the preparations can be used for more than six months.
If the experimenter does not wish to prepare his/her own human metaphases, fairly good spreads prepared specifically for CGH experiments
can be purchased (Vysis, Inc. Downers Grove, IL). However, since methods for preparing mouse metaphases are not generally available, we provide a method here that we use for CGH.
Materials
Reagents - Complete medium: Sterile fIlter 450 ml Dulbecco's modified Eagle
medium (F-12/H-21 0:1),50 ml fetal calf serum, 7.5 ml 1M HEPES,
5 ml 0.2 M glutamine, 5 ml pen/strep (10,000 U/ml penicillin and 10,000
mg/ml strepomycin).
- Hank's Balanced Salt Solution, Calcium and Magnesium Free