Staining chromosomes (G-Banding).ppt

Staining chromosomes (G-Banding) 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. 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. Procedure: 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. Dip oven-dried slides in the trypsin solution for 3-7 seconds. Rinse slides in water. 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. Rinse slides in water and air dry. Check the slides using a Micoscope, Low magnification lens , high magnification lens ,100X oil objective, brightfield (Xylene ). Cedar wood oil Over-trypsinized chromosomes appear fuzzy; somewhat difficult to recognize exact bands. Under-trypsinized chromosomes will have indistinct bands, decreased contrast; very difficult or impossible to determine bands. Adequately trypsinized chromosomes will show telomeres not overly digested and G-Bands will appear sharp and in contrast. G banding song 一秃二蛇三蝶飘，四像鞭炮五黑腰；六号像个小白脸，七盖八下九苗条，十号长臂近带好，十一低来十二高；十三、四、五一二一；十六长臂缢痕大，十七长臂带脚镣，十八白头肚子饱；十九中间一点腰，二十头重脚飘飘；二十一好象黑葫芦瓢，二十二头上一点黑；X染色一担挑，Y染色长臂带黑脚。