Heterochromatin is condensed throughout interphase and metaphase and is therefore relatively constant in size. They are usually located near the mitotic grains or appear as "clumps" on the chromosome arms, and these chromatin are mainly visualized by the technique of C-banding, which is short for C-banding, Ba(OH)2, and Giemsa. The fundamental characteristic of C-banding is that it selectively extracts DNA from the chromosome arms, but it is highly resistant in the C-banding region, where it retains a large portion of the DNA, and is seen with good results after Giemsa staining. Giemsa staining shows good C-banding.
Operation method
basic program
Principle
Heterochromatin is condensed throughout interphase and metaphase and is therefore relatively constant in size. They are usually located near the mitotic grains or in "clumps" on the chromosome arms, and these chromatin are mainly presented by the technique of C-banding, hence the name C-banding. CBG is shorthand for C-banding, Ba(OH)2, and Giemsa, and the fundamental characteristic of C-banding is that it selectively extracts DNA from the chromosome arms, but is highly resistant in the C-banding region, retaining most of the DNA. that still retains most of the DNA, and C-banding with good results is visible after Giemsa staining. The process of DNA extraction is shaped by three successive operations. First, acid treatment depurinates the DNA without breaking the DNA backbone; secondly, hot alkali treatment denatures the DNA and promotes secondary DNA solubilization; and finally, hot salt solution breaks the DNA backbone and dissolves the fragments into solution. As a result, the final Giemsa dye shows a different distribution of DNA. In good C-banded specimens, the euchromatin shows only the general appearance of the intermediate chromosomes, and the structural heterochromatin regions are very darkly colored.C-banding is particularly important in examining the heterogeneous regions of chromosomes 1, 9, 16, and especially the Y chromosome.
Materials and Instruments
Chromosome specimens Move I. Supplies and reagents0.2N HCl, 5% Ba(OH)2, 2×SSC, Giemsa staining solution. Constant temperature water bath, staining cylinder. The rest was the same as peripheral blood chromosome preparation. For more product details, please visit Aladdin Scientific website.
HCl Ba(OH)2 SSC Giemsa staining solution
Thermostatic water bath Staining vat Microscope
II. Operational steps
Basic Program
1. Acid treatment: Routinely prepared chromosome specimens (unstained) are treated with 0.2 N HCl (room temperature) for 15-30 minutes. Wash with water.
2. Alkali treatment: Immerse in a 5% aqueous Ba(OH)2 solution pre-warmed to 56°C for 10 minutes. Wash in water.
3. Hot salt treatment: Place in 2 x SSC solution (67°C) for 60-90 minutes. Wash with water.
4. Staining: l:10 Giemsa stain for 10-5 minutes. Wash with water. Air dry.
5. Mirroring.
Alternatives:
1. Acid treatment: Routinely prepared chromosome specimens (unstained) are treated with 0.2 N HCl (room temperature) for 60 minutes. Wash in water.
2. Alkali treatment: Immerse in 1% Ba(OH)2 aqueous solution (50°C) for 15-20 seconds. Wash with water.
3. Hot salt treatment: Place in 2 x SSC solution (60°C) for 90 minutes. Wash with water three times.
4. Staining: l:10 Giemsa stain for 10 minutes. Wash with water. Air dry.
5. Mirroring.