G- & C-bands protocol

Muriel T. Davisson

Solutions needed

      2XSSC = 8.8 g NaCl

      4.4 g trisodium citrate

      Make up to 500 ml with distilled water

      Trypsin-Giemsa solution

      1.0 ml Karyomax Giemsa (Gurr's Improved R66 #10092-013)*

      45 ml Gurr pH 6.8 phosphate buffer (#10582-013)*

      4 drops 0.0125% trypsin (Sigma 2.5% solution #T2021)

      0.9% NaCl = 0.9 g NaCl in 100 ml distilled water

* Invitrogen (Gibco) 1-800-828-6686

(Davisson, Akeson. Cytogenet Cell Genet 1987; 45:70-74)

G-bands

1. Make air-dried preparations by dropping small droplets of cell suspension on the slides and blowing dry. Bands are sharper if preparations are aged 7-10 days at room temperature, but this is not essential.
2. Incubate slides in Coplin jars (5-6 per jar) in 2XSSC at 60-65°C for 1 1/2 hrs.
3. Transfer all slides to 0.9% NaCl at room temperature. Then rinse each slide in fresh NaCl and drain. Thorough rinsing is critical.
4. Stain 4-6 minutes in trypsin-Giemsa solution (below). Remove the metallic film that forms on the stain surface with a cotton ball before placing slides in Coplin jar or float the film off with running water before removing slides.
5. Transfer all slides in jar to fresh buffer (1:1 buffer:dist water).
6. Rinse slides individually in 2 changes of buffer (1:1 buffer:dist water). [Thorough rinsing is critical.] Shake off excess liquid and blow dry with an air jet.

C-bands

1. Previously aged and G-banded slides are destained in methanol (10 minutes).
2. The above G-band method is repeated to show the centromeric heterochromatin. (The staining time may need to be increased to 10-12 minutes depending on the age of the slides - older slides need to be stained longer.)

(Akeson, Davisson. Cytogenet Cell Genet 1991; 57:217-220)

Simultaneous G- and C-banding

1. Incubate freshly made slides in oven at 65°C for 17-18 hours.
2. Follow the above G-band method (steps 2-6).
3. The staining time may need to be increased to 8-10 minutes.
4. This method visualizes both the darkly staining centromeric heterochromatin and the G-band pattern of the chromosomes.

Unbanded Giemsa stained chromosomes

1  Stain mitotic cells 5 minutes in 4% Gurr Giemsa in Gurr buffer (for bone marrow).

• 2 ml Karyomax Giemsa (Gurr's Improved R66 solution #10092-013)
• 48ml Gurr buffer pH 6.8 (buffer tabs #10582-013)
• Rinse under cool running tap water followed by several rinses of distilled water.

2  Stain meiotic cells 5 minutes as above except when doing testis preparations use Fisher stain.

• 2ml Fisher Giemsa (#SG28-475)
• 48ml PO4 buffer pH 7.0 (#SB108-20) or Gurr buffer, pH 6.8(# 10582-013)
• Rinse under cool running tap water followed by distilled water.

Quick G-banding

1. Place slides in 60-65°C incubator overnight.
2. Stain in 2.2% Giemsa with 4 qtts 0.0125 trypsin (1mlGiemsa / 45ml Gurr buffer)
3. Leave 4-6 mins

Quick staining

1. 1ml Gurr Geimsa
2. 45ml Gurr buffer
3. Leave 4-5 mins