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 细胞凋亡-DNA Ladder抽提试剂盒

  产品编号C0007
  产品包装: 50次
  产品价格: 199.00元
产品编号 产品名称 产品包装 产品价格
C0007 细胞凋亡-DNA Ladder抽提试剂盒 50次 199.00元

      碧云天生产的细胞凋亡-DNA Ladder抽提试剂盒,是针对细胞凋亡过程中产生的核小体间DNA链断裂而设计的。可以非常有效地抽提最小片段为180-200bp的DNA ladder,同时又可以抽提到50kb以上的基因组DNA。
      DNA ladder也称DNA fragmentation,是细胞凋亡的一个重要指标。通常观察到DNA ladder,就可以判定细胞发生了凋亡。
      本试剂盒足够抽提50个细胞或组织样品。
包装清单:

产品编号 产品名称 包装
C0007-1 样品裂解液 30ml
C0007-2 蛋白酶K 130μl
C0007-3 10M 醋酸铵 6ml
C0007-4 TE 6ml
说明书 1份

保存条件:
      -20℃保存,一年有效。10M 醋酸铵和TE也可以室温保存。
注意事项:
      需自备Tris平衡苯酚、氯仿和无水乙醇。
      本产品仅限于专业人员的科学研究用,不得用于临床诊断或治疗,不得用于食品或药品,不得存放于普通住宅内。
      为了您的安全和健康,请穿实验服并戴一次性手套操作。

使用说明:
1.样品收集
a)对于组织样品
切下组织,并剪切成小块,置液氮中冻结,研碎或捣碎。或直接冰浴上匀浆。
b)对于贴壁细胞
胰酶消化后,PBS或生理盐水洗一次,1000-2000g离心1-2分钟,弃上清,收集细胞。
c)对于悬浮细胞
1000-2000g离心1-2分钟,弃上清,收集细胞。
2.DNA ladder抽提
a)每1毫升样品裂解液中加入5微升蛋白酶K,混匀。
b)对于上述收集好的样品,每5毫克组织或者106个细胞中加入500微升添加了蛋白酶K的样品裂解液,Vortex混匀,充分裂解组织或细胞。
c)50℃水浴消化过夜(通常12-20小时皆可)。
d)加入500微升Tris平衡苯酚(pH8.0)。
e)Vortex剧烈混匀,使有机相和水相充分混合,以达到抽提效果。4℃,12,000g离心5分钟。
f)缓慢吸出上层水相至另一洁净离心管中。注意勿触及中间层,中间层通常含有变性的蛋白等,并用等体积Tris平衡酚再抽提一次(同步骤e)。
g)缓慢吸出上层水相至另一洁净离心管中。注意勿触及中间层,中间层通常含有变性的蛋白等,并用等体积氯仿再抽提一次(同步骤e)。
h)慢慢吸出约300微升上清液,加入60微升10M醋酸铵和600微升无水乙醇,颠倒数次混匀,此时可见DNA沉淀产生。-20℃冻存1小时,以充分沉淀小片段DNA。冻存过夜或-70℃冻存效果更佳。
i)4℃,12, 000g 离心10分钟,弃上清。
j)加入600微升70%乙醇,轻轻颠倒约2次。4℃,12, 000g 离心10分钟,小心吸去上清。
注意:70%乙醇洗涤的时候,千万注意避免损失一些细小的DNA沉淀,这些沉淀中大部分是你所需的DNA ladder。
k)尽量吸除残余的乙醇,待看不到明显的液体时,立即加入50-100微升TE溶解DNA。
注意:不可过分干燥基因组DNA沉淀,否则会极难溶解。如果发现DNA沉淀难以溶解,可以在4℃用摇床缓慢摇动过夜,以溶解DNA沉淀。
l)取部分抽提得到的DNA,1%琼脂糖凝胶电泳分析。如果细胞发生凋亡,就可以观察到典型的DNA ladder。电泳时一定要注意换用新鲜配制的电泳液,DNA凝胶也要用新鲜配制的电泳液配制并新鲜配制后使用。电泳时为获取最佳的电泳效果使ladder充分分开,电泳速度宜适当慢一些,凝胶宜适当长一些,而加样孔宜更加扁平一些。选取适当较薄的梳齿,往往会获得更好的ladder电泳效果。
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