2. Hypotonic treatment causes a swelling of the cells; the optimal time of treatment varies for different cell types and must be determined empirically (membrane; chromosome) 3. If the slides are not made the same day as the harvest, fill the tube with freshly prepared fixative, tighten the cap, and I Chromosome analysis G-banding is technique used in cytogenetics to produce a visible karyotype by staining condensed chromosomes. The metaphase chromosomes are treated with trypsin(to partially digest the protein) and stained with Giemsa. Dark bands that take up the stain are strongly A, T rich To stain metaphase chromosomes with Giemsa to elicit a banding pattern throughout the hromosome arms, designated G-Bands. This G-Banding technique requires a chromosomal pretreatment step of trypsin to induce chromosome bands 2. Procedure 1. Dissolve 25mg trypsin in 50 ml 0.85% NaCl/ sodium chloride( trypsin solution ) 37 C, add 2 drops of.4% Phenol-sulfonphthalein, adjust pH to 6.4-6.6 with 5%NaHCO3/sodium bicarbonate 2. Dip oven-dried slides in the trypsin solution for 3-7 seconds 3. Rinse slides in water 4. Use a graduated tube to mix 0.5 ml Giemsa and 5 ml PBS buffer just prior to staining. Stain the slides for 8-10 minutes in a coplin staining ja 5. Rinse slides in water and air d 6. Check the slides using a Micoscope, Low magnification lens, high magnification lens, 100X oil 7. Cedar wood oi 1.Over-trypsinized chromosomes appear fuzzy; somewhat difficult to recognize exact bands 2. Under-trypsinized chromosomes will have indistinct bands, decreased contrast; very difficult or impossible to determine bands 3. Adequately trypsinized chromosomes will show telomeres not overly digested and g-Bands will appear sharp and in contrast2. Hypotonic treatment causes a swelling of the cells; the optimal time of treatment varies for different cell types and must be determined empirically. (membrane; chromosome) 3. If the slides are not made the same day as the harvest, fill the tube with freshly prepared fixative, tighten the cap, and store the suspension at -20°C. II Chromosome Analysis G-banding is technique used in cytogenetics to produce a visible karyotype by staining condensed chromosomes.The metaphase chromosomes are treated with trypsin (to partially digest the protein) and stained with Giemsa. Dark bands that take up the stain are strongly A,T rich. 1.Purpose: To stain metaphase chromosomes with Giemsa to elicit a banding pattern throughout the chromosome arms, designated G-Bands. This G-Banding technique requires a chromosomal pretreatment step of trypsin to induce chromosome bands. 2.Procedure: 1.Dissolve 25mg trypsin in 50 ml 0.85% NaCl / sodium chloride (trypsin solution ). 37 ℃, add 2 drops of 0.4% Phenol-sulfonphthalein, adjust pH to 6.4-6.6 with 5%NaHCO3/sodium bicarbonate. 2.Dip oven-dried slides in the trypsin solution for 3-7 seconds. 3.Rinse slides in water. 4.Use a graduated tube to mix 0.5 ml Giemsa and 5 ml PBS buffer just prior to staining. Stain the slides for 8-10 minutes in a coplin staining jar. 5.Rinse slides in water and air dry. 6.Check the slides using a Micoscope, Low magnification lens , high magnification lens ,100X oil objective, brightfield (Xylene ). 7.Cedar wood oil 3. Analysis 1.Over-trypsinized chromosomes appear fuzzy; somewhat difficult to recognize exact bands. 2.Under-trypsinized chromosomes will have indistinct bands, decreased contrast; very difficult or impossible to determine bands. 3.Adequately trypsinized chromosomes will show telomeres not overly digested and G-Bands will appear sharp and in contrast