G- & C-bands protocol

Muriel T. Davisson

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)

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.
Incubate slides in Coplin jars (5-6 per jar) in 2XSSC at 60-65°C for 1 1/2 hrs.
Transfer all slides to 0.9% NaCl at room temperature. Then rinse each slide in fresh NaCl and drain. Thorough rinsing is critical.
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.
Transfer all slides in jar to fresh buffer (1:1 buffer:dist water).
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.

Previously aged and G-banded slides are destained in methanol (10 minutes).
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)

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