小鼠ES细胞核型分析

寒山拾得

G Banding
" I2 r6 p+ S" [  C0 i" P  A: q: K. L10. Place suitably aged slides (see note below) in 2X SSC in a Coplin staining jar with a lid in a water bath at 60°C-65°C for 1.5 h. Then cool the slides to room temperature by running tap water over the closed jar. Transfer the slides to 0.85% (w/v) NaCl at room temperature for 5 min.* x2 b% D# N) A% x% A# K% o
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Before G banding, slides should be “aged” for between 3 and 21 d by leaving them in a closed box at room temperature. Fresh slides give poor G-band resolution. Maximum G-band resolution is achieved at ~10 d after slide preparation. Beyond this time, resolution slowly decreases until, after several weeks in storage, the chromosomes either fail to band and stain uniformly or show “pseudobands” that are not significant to the standard idiogram.& l0 g5 y6 W+ i* o6 U  H4 Z
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11. Drain the slides by touching them onto filter paper. Place them on a flat surface and flood the chamber with 0.025% trypsin in 0.85% NaCl for 15-20 sec.
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The trypsin exposure time is critical: Underexposure preserves chromosome morphology but gives poorly differentiated bands, and overexposure distorts morphology and eliminates most of the bands. Optimum trypsin times are known to vary among laboratories. A test slide should be treated for the minimal suggested time of 15 sec to establish the best treatment time for the rest of the slides.: R8 t- D0 U4 @9 J3 {( q" y7 n. K
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In the laboratory of E.P. Evans, the optimal trypsin exposure time has been established as between 15 and 20 sec for mouse chromosomes, irrespective of the source of the mitotic cells.
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12. Stop tryptic activity by placing the slides back into 0.85% NaCl. Then rinse slides in phosphate buffer (pH 6.8) and stain in fresh Giemsa stain in 5 mM phosphate buffer (pH 6.8). After 10 min in the stain, monitor wet slides under low-power, bright-field microscopy (160X) for staining intensity. Because, upon drying, wet slides gain contrast, care should be taken not to overstain the cells as this will reduce G-band differentiation. If necessary, repeat staining until adequate results are achieved and then quickly rinse slides in phosphate buffer (pH 6.8) and blow-dry with a current of cool air.
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13. Examine unmounted slides with a 100X oil-immersion lens.
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The majority of modern, readily available immersion oils that are declared “PCB-free” also have the unfortunate property of removing Giemsa stain after a few hours of exposure. Although direct viewing of slides under an oil immersion lens gives a higher optical resolution, it is wise to mount slides if they are to be kept.

iseeyou1210

ES cell 核型分析一般就是看染色体的数目是不是和标准的一致。做G带，也看不出什么更deep的change，而且小鼠的染色体还真的不好认。2 }/ h2 G9 J7 T
good luck！