88
MORIMASA MATSUTA AND MAYUMI MATSUTA
Hybridization
mix preparation
• Distorted cell morphology
- Specimens were not dried during preparation: Increase
drying time to at least overnight at room temperature,
and then age slides at least 24 hours at room temperature.
- Specimen slides not thoroughly dry prior to immersion in
denaturation solution: Dehydrate slides through a series of
1 minute ethanol washes (7S%, 80%, 100%), and air dry.
- Specimen over-digested: If using proteolytic treatment
with proteinase K or pepsin, use the enzymes at lower concentration. Ensure temperature of denaturation solution is
70-74°C, and decrease the temperature to 70°C.
Subprotocol 2
Protocol for Rapid Procedure
Procedure
Note: Heat or enzymatic treatment may induce loss of adherence
of cells and tissue sections to glass slides. This may be overcome
by using aminoalkylsilane-coated glass slides.
Note: Prior to use, thaw reagents at ambient temperature, then
centrifuge each tube for 2-3 secs using a standard bench-top microcentrifuge.
Prepare three Coplin jars: pour SOml 100% ethanol into one
jar: SOml 8S% ethanol into another, and SOml 70% ethanol into
the last.
Bring Coplin jars containing the denaturation solution and
OAX SSC to ambient temperature. Place jars in a 74°C water
bath approximately 30 mins prior to use to attain the solution
temperature. Measure the temperatures of the solution inside
the Coplin jar.
1. Add the following to a microcentrifuge tube at ambient temperature:
- htl of master mixture
- 1)l1 carrier DNA (SOO)lg/ml)
MORIMASA MATSUTA AND MAYUMI MATSUTA
Hybridization
mix preparation
• Distorted cell morphology
- Specimens were not dried during preparation: Increase
drying time to at least overnight at room temperature,
and then age slides at least 24 hours at room temperature.
- Specimen slides not thoroughly dry prior to immersion in
denaturation solution: Dehydrate slides through a series of
1 minute ethanol washes (7S%, 80%, 100%), and air dry.
- Specimen over-digested: If using proteolytic treatment
with proteinase K or pepsin, use the enzymes at lower concentration. Ensure temperature of denaturation solution is
70-74°C, and decrease the temperature to 70°C.
Subprotocol 2
Protocol for Rapid Procedure
Procedure
Note: Heat or enzymatic treatment may induce loss of adherence
of cells and tissue sections to glass slides. This may be overcome
by using aminoalkylsilane-coated glass slides.
Note: Prior to use, thaw reagents at ambient temperature, then
centrifuge each tube for 2-3 secs using a standard bench-top microcentrifuge.
Prepare three Coplin jars: pour SOml 100% ethanol into one
jar: SOml 8S% ethanol into another, and SOml 70% ethanol into
the last.
Bring Coplin jars containing the denaturation solution and
OAX SSC to ambient temperature. Place jars in a 74°C water
bath approximately 30 mins prior to use to attain the solution
temperature. Measure the temperatures of the solution inside
the Coplin jar.
1. Add the following to a microcentrifuge tube at ambient temperature:
- htl of master mixture
- 1)l1 carrier DNA (SOO)lg/ml)