Giemsa Banding (“GTG” Banding)

Giemsa banding (GTC banding) procedure

Banding of chromosome with enzymes and stains is essential to identifying normal and abnormal chromosome structures.

Reagents

  1. Gurrs 6.8 buffer tablets: (Cat. No. 10582013) one tablet (100 mL, 500 mL, 1 liter size) dissolved in distilled water
  2. 0.9% sodium chloride: 4.5 grams NaCl made up to volume of 500 mL distilled water
  3. Gurrs stain (R66) Giemsa:
    • 3.0 mL Giemsa stain (Cat. No. 10092013) 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%:
    • 2.5 mL Trypsin (10X) (Cat. No. 15090046)
    • 49.5 mL 0.9% NaCl
    • Mix in Coplin jar

Equipment

  • Coplin jars or larger containers
  • Forceps
  • Coverslips (24 mm X 50 mm)
  • 5 mL serological pipets (Cat. No. 170355)
  • 1 mL serological pipets (Cat. No. 170353)
  • 50 mL graduated cylinder
  • 50°C warming oven 

Procedure

  1. Six Coplin jars are used for the banding sequence
    1. First jar—0.125% trypsin/0.9% NaCl mixture
    2. Second jar—0.9% NaCl for rinsing
    3. Third jar—0.9% NaCl for rinsing
    4. Fourth jar— Gurrs Giemsa stain (R66) mixed with Gurrs 6.8 buffer and acetone
    5. Fifth jar— Gurrs 6.8 buffer for rinsing
    6. Sixth jar— Gurrs 6.8 buffer for rinsing
  2. 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 sec or as long as 2 min, depending on the activity level of the trypsin being used.
  3. After the trypsin time has elapsed, the slide is removed and rinsed by sequential dipping into the 0.9% NaCl rinsing jars.
  4. 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.
  5. 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.
  6. The slide is removed from the last rinse and air dried and coverslipped with Cytoseal 60. It is allowed to dry in the oven (50°C) after which it is ready for metaphase scanning under the microscope.

References

  1. Breg, RW, Karyology and Chromosome Banding of Cultured Cells, Dec. 6-10, 1976 Lake Placid, NY.
  2. Sun, NC, Ehy, Chu, CC Chang (1976) Staining Method for Banding Patters Human Mitotic Chromosomes. MCN 14-26.
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