G Banding

Procedure:

1. De-stain slides for 10 minutes in 95% ethanol

2. Place slide in PBS ( phosphate Buffer Solution) and incubate for 10 MINUTES AT 56° c

3. Treat silde with 0.22 mL of 25 % trypsin, 2.5 mL of methanol and 0.22 mL of stock giemsa and 6.5 mL 0f PBS with a pH of 7.4.

4. Flood slide with stain solution for 15 minutes. Rinse with water and air dry

5. View slide under bright field, oil immersion microscope.

Our Results:

Click Here for Image Click Here for Image

Variations for G Banding:

1. Mix giemsa stain solution without trypsin.

2. Put solution in -75° freezer for 25 minutes

3. Add 3 drops of cell suspention to slide and blow in one direction and allow to dry

4. Put side in PBS solution at 56°C for 10 minutes.

5. Directly add 25% trypsin to slide for 10 minutes.

6. Add stain without the trypsin for 10 minutes.

Variation II

1. Mix 2.5 mL of methanol and 0.22 mL of stock giemsa and 6.5 mL 0f PBS with a pH of 7.4.

2. Repeat steps in variation 1-3 above.

3. Place trypsin directly onto dried slide for 30 seconds.

4. Rinse trypsin off in PBS at 60° C

5. Treat slide with giemsa mix for 9 minutes.

6. Rinse silde in water and dry in 60°C oven.

7. View under bright field, oil immersion microscope.

Variation III

1. Prepare side as in variation steps 1-3 above.

2. Mix 2.5 mL of methanol and 0.22 mL of stock giemsa.

3. Prepare trypsin solution – 0.25mL 2.5% trypsin; 6.5 mL 0f PBS with a pH of 7.4.

4. Treat 2 sides with trypsin solution one for 25 minutes and another for 35 minutes.

5. Stain slides with dye solution for 25 minutes.

6. Rinse with distilled water

7. View under bright field, oil immersion microscope.

Variation IV– Cold Dye Technique:

1. Prepare side as in variation steps 1-3 above.

2. Place trypsin in refrigerator at 4°C for 20 minutes.

3. Add giemsa mixture for 7.5 minutes.

4. Rinse with water.

5. Place slide in 60°C oven to evaporate water.