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GTL-BANDING (Using Trypsin)
I. Purpose:
A. Identification of individual chromosomes and their structural anomalies.
II. Procedure:
A. Harvest the cell culture according to the standard procedure. Dry the slides in air at room temperature for at least 24 hours. Optimal age of slides is 24 hours - one week, however, slides can be stained earlier or later, though band quality is usually poorer.
B. Bake slides for 15-45 minutes at 90 - 95 C.
C. Prepare 3 coplin jars.
D. Staining can be done either in a coplin jar or horizontally on a slide tray. For both the stain solution must be prepared fresh. For the coplin jar use 45 ml Gurr Buffer pH 6.8 and 15 ml Leishman stain. For horizontal staining prepare 3 ml Gurr Buffer pH 6.8 and 1 ml Leishman stain in a tube for each slide. Layer the stain carefully over the Gurr buffer and mix just prior to use.
- (1) 50 ml Isotonic buffer + 0.2 ml trypsin working solution
- (2) 50 ml Isotonic buffer
- (3) 50 ml Gurr Buffer pH 6.8
E. Use 1 slide as a trial to determine the optimal trypsin and stain times for each patient .
F. Place slide in coplin jar #1 to trypsinize, rinse briefly in coplin jar #2, then rinse briefly in coplin jar #3. Drain slide.
G. Stain either in coplin jar or horizontally on slide tray.
H. Rinse briefly under cold running tap water.
I. Wipe the back of the slide dry and dry the slide by air jet or by air drying.
J. Examine slide under oil immersion to assess the quality of the banding. If the chromosomes are uniformly stained but no bands are seen, the trypsin time was too short. If the chromosomes are swollen and indistinct, the trypsin time was too long. In either case, repeat the trial procedure making appropriate changes in trypsin time. Several trials may be necessary until a sharp pattern of bands is achieved. Overstaining or understaining with Leishman may also result in a preparation of poor quality. Once the optimal trypsin and staining times have been determined, prepare slides for analysis.
III. Solutions:
Gurr Buffer: Dissolve 1 Gurr buffer tablet pH 6.8 (1 liter solution) in 1 liter dH2O.
Isotonic Buffer: Dissolve 36 grams NaCl, 8 Gurr buffer tablets pH6.8 (100 ml solution), and 4 Gurr Buffer tablets pH 7.0
(1 liter solution) in 3950 ml dH2O. Adjust pH to 7.0 with 2.5 ml 2M NaOH. Bring volume to 4 liters with dH2O.
Leishman Stain: Dissolve 1 gram Leishman stain in 500 ml Methanol. Incubate at 37 C overnight. Filter through Whatman paper qualitative grade 1.
Trypsin working solution: Dissolve 0.1 gram Trypsin 1:250 in 4 ml sterile dH2O. Store frozen.
IV. Reagents:
Gurr Buffer Tablets: BIO/MEDICAL SPECIALTIES cat# 33193-2P. Buffer Tablets pH 6.8 (1 liter of solution) BPS 50 tablets/bottle.
Gurr Buffer Tablets: BIO/MEDICAL SPECIALTIES cat# 33193-2D. Buffer Tablets pH 6.8 (100 ml of solution) BPS 50 tablets/bottle.
Gurr Buffer Tablets: BIO/MEDICAL SPECIALTIES cat# 33200-2U Buffer Tablets pH 7.0 (1 liter of solution) BPS 50 tablets/bottle.
Leishman Stain: SIGMA cat. # L-6254 Harleco Leishman Stain, 25 gram/bottle. Store in dark.
Methanol: FISHER cat.# A412-500 Methyl alcohol anhydrous AR (ACS)(Absolute) Acetone free, 500 ml bottle.
Trypsin: GIBCO cat# 27250-042. Trypsin 1:250,
25 gram/bottle.