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大马哈鱼精子DNA的制备

标签: 大马哈鱼 精子DNA

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1) Use Pharmacia #27-4564-01. With clean flamed scissors and forceps, weigh 0.25 g/50 ml conical and add 50 ml/conical of 0.02 M Tris, pH 7.6. Allow to dissolve over several days at 4℃.

2) Vortex. Draw up into a 20 or 30 ml syringe. Add a 25 guage needle and shear DNA by squirting through needle into fresh 50 ml conicals at 15 ml/conical. To this add 0.9 ml/conical of 5 N NaCl.

3) Boil in water bath (95 - 100℃) for 20 min. Cool in ice water. Vortex and then add 1 drop (from a standard transfer pipet) of 1.2 N HCl (concentrated HCl diluted 1/10) to neutralize.

4) Add two volumes of 100% ethanol, let stand for 20 min on ice, then spin for 10 min in the Beckman GS-6R at 2500 rpm (4℃). Pour off the supernatant, let dry for 5 min, then add 15 ml/tube of 0.02 M Tris, pH 7.6. Vortex and let dissolve for several days at 4℃. Determine OD260 of a diluted aliquot to check concentration (1 OD260 = 50 µg DNA/ml), then store at -20℃.

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