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
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)
Simultaneous G- and C-banding
- 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.
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.
- Place slides in 60-65°C incubator overnight.
- Stain in 2.2% Giemsa with 4 qtts 0.0125 trypsin (1mlGiemsa / 45ml Gurr buffer)
- Leave 4-6 mins
- 1ml Gurr Geimsa
- 45ml Gurr buffer
- Leave 4-5 mins