392
ILSE CHUDOBA and GABRIELE SENGER
GTG-banding
One ampoule of the lyophilized Bacto trypsin (Difco) is dissolved in 10 ml
sterile distilled water. Store 100 III aliquots at -20°C.
Banding procedure is carried out in 50 ml sterile plastic centrifuge tubes.
Altogether 4 tubes are necessary which should be prepared as follows:
- 35 ml phosphate buffer, pH 6.88 (Merck)
- 35 ml phosphate buffer, pH 6.88 (Merck) and 100 III Bacto trypsin
- 35 ml phosphate buffer, pH 6.88 (Merck) and 1.5 ml Giemsa (Merck)
- 50 ml sterile distilled water
1. I-min incubation in tube 1
2. I-min incubation in tube 2 (trypsin digestion)
3. 2-min incubation in tube 3 (staining of the chromosomes)
4. rinse (tube 4) coverslips briefly and air dry
Pre-incubation in tube 1 is optional, but was observed to improve GTGbanding of freshly spread metaphases. The incubation times of trypsin
digestion (tube 2) and Giemsa staining (tube 3) is variable and have to
be adjusted to laboratory standards.
Microdissection
To facilitate handling of the fragile coverslips, a metal plate with a rectangular hole in the middle (-5.5 x 5.5 cm) is used as a carrier.
Prepare PeR-tubes containing 4 III of reaction mix A.
Reaction mix A
Conc. stock
Actual conc.
Final conc.
H2O
Sequenase buffer
5x
1.25
Ix
Proteinase K
14 mg/ml"
2.5 mg/ml
2 mg/ml
" Varies with lot no.
Volume required
11.4 III
5.0 III
3.6 III
conc. stock: concentration of the stock solution
actual conc.: concentration resulting for the actual reaction mix A
final cone.: concentration resulting after adding reaction mix B