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Introduction - Procedure for Staining
Banding of chromosome with enzymes and stains is essential to identifying normal and abnormal chromosome structures.
Equipment
Reagents:
1. Gurrs 6.8 buffer tablets (Gibco Cat #10582-013)
one tablet dissolved in 1L distilled water
2. 0.9% NaCl
4.5 grams NaCl made up to volume of 500 mL distilled water
3. Gurrs Giemsa stain (R66)
3.0 mL Giemsa stain (GIBCO Cat #10092-013) added to 48.5 Gurrs 6.8 buffer (unless otherwise stated on C of A)
1 mL acetone
Mix in coplin jar
4. Trypsin 2.5% (10X) (GIBCO Cat #15090-046)
2.5 mL trypsin
49.5 mL 0.9% NaCl
Mix in coplin jar
Equipment
- coplin jars or larger containers
- forceps
- coverslips (24mm X 50mm)
- 5 mL serological pipets
- 1 mL serological pipets
- 50 mL graduated cylinder
- 50C warming oven
Reagents:
1. Gurrs 6.8 buffer tablets (Gibco Cat #10582-013)
one tablet dissolved in 1L distilled water
2. 0.9% NaCl
4.5 grams NaCl made up to volume of 500 mL distilled water
3. Gurrs Giemsa stain (R66)
3.0 mL Giemsa stain (GIBCO Cat #10092-013) added to 48.5 Gurrs 6.8 buffer (unless otherwise stated on C of A)
1 mL acetone
Mix in coplin jar
4. Trypsin 2.5% (10X) (GIBCO Cat #15090-046)
2.5 mL trypsin
49.5 mL 0.9% NaCl
Mix in coplin jar
Protocol
- Six coplin jars are used for the banding sequence.
- A slide is placed in the first coplin jar containing the trypsin/NaCl mixture for a prescribed amount of time. This time may be as short as 10 seconds or as long as 2 minutes, depending on the activity level of the trypsin being used.
- After the trypsin time has elapsed, the slide is removed and rinsed by sequential dipping into the 0.9% NaCl rinsing jars.
- The slide is then placed in the staining jar containing the Gurrs stain and buffer for 5 minutes. This time may vary somewhat, depending on the strength of the stain used.
- After the staining time has elapsed, the slide is removed from the jar and rinsed by sequential dipping into the two Gurrs buffer rinsing jars.
- The slide is removed from the last rinse and air dried and coverslipped with Cytoseal 60. It is allowed to dry in the oven (50C) after which it is ready for metaphase scanning under the microscope.
1st: 2.5% trypsin/0.9% NaCl mixture
2nd: 0.9% NaCl for rinsing
3rd: 0.9% NaCl for rinsing
4th: Gurrs Giemsa stain (R66) with Gurrs 6.8 buffer and acetone
5th: Gurrs 6.8 buffer for rinsing
6th: Gurrs 6.8 buffer for rinsing
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