Giemsa Banding (“GTG” Banding)

Introduction - Procedure for Staining

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

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
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Protocol

  1. Six coplin jars are used for the banding sequence.
  2.         
              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

  3. 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.

  4. After the trypsin time has elapsed, the slide is removed and rinsed by sequential dipping into the 0.9% NaCl rinsing jars.

  5. 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.

  6. 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.

  7. 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.
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Ordering Information

Catalog # Name Size List Price (USD) Qty
10582013 Gurr Buffer Tablets 50 x 1 L 121.00
10092013 KaryoMAX® Giemsa Stain Solution 100 mL 45.50
15090046 Trypsin (2.5%), no phenol red 100 mL 28.80

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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