CGH 切口平移:制备试剂

CGH 切口平移:制备试剂

 

0.2 mM SpectrumGreen 或 SpectrumRed dUTP

将 10 µL 1 mM dUTP 添加至 40 µL 无核酸酶水中。

 

0.1 mM dTTP

将 10 µL 0.3 mM dTTP 添加至 20 µL 无核酸酶水中。

 

0.1 mM dNTP 混合物

将 0.3 mM dATP、0.3 mM dCTP 和 0.3 mM dGTP 各 10 µL 混合在一起。

 

提取的基因组 DNA

在 Tris-EDTA (10 mM Tris、1 mM EDTA,pH 8.5) 缓冲液中制备 0.2 µg/µL 至 1 µg/µL 基因组 DNA 提取溶液。

 
 
 
 
CGH 切口平移:测定程序

 

此程序可标记大约 1 µg 提取的基因组 DNA。这足以进行五次 CGH 杂交。

  1. 将微量离心管放在冰上,让离心管冷却。
  2. 按列出的顺序将这些组分加入离心管中:
    cgh-nick-translation-assay-procedure.gif
  3. 简短地涡旋离心管。
  4. 在 15 °C 下孵育 2 - 4 小时。
  5. 通过在 70 ℃ 水浴中加热 10 分钟来停止反应。
  6. 在冰上冷却。 

确定探针大小

确定探针大小是 CGH 程序中必不可少的一部分。有关制备和进行琼脂糖凝胶的详细说明,请参见 (1) Maniatis T, Fritsch EF, and Sambrook J. Gel electrophoresis of DNA.In: Molecular cloning: a laboratory manual.2nd ed. Cold Spring, NY: Cold Spring Harbor Laboratory; 1989 or (2) Ausubel FM, ed. Preparation and Analysis of DNA.In: Current Protocols in Molecular Biology.New York: Greene Publishing Associates and John Wiley & Sons, 1989.

  1. 通过向 100 mL TAE 缓冲液中加入 1 g 琼脂糖来制备 1% 琼脂糖凝胶。在微波炉中加热溶液,直至琼脂糖溶化。此容量足以制作三块微型凝胶。
  2. 将琼脂糖溶液冷却至 55 °C 并加入 10 µL EtBr(最终浓度为 0.01%(体积比))。
  3. 将溶化的琼脂糖倒入装有梳板的微型凝胶(10 厘米 x 6.5 厘米)装置中。使琼脂糖冷却固化。
  4. 将足够的 TAE 电泳缓冲液倒入微型凝胶装置中以覆盖凝胶约 1 mm。
  5. 取 9 µL 含有缺口平移 DNA 的反应混合物,并加入 1 µL 凝胶上样缓冲液。
  6. 在一条泳道中加载缺口平移样本,在另一条泳道中加载 DNA 大小标记样本进行电泳以确定探针的大小。
  7. 以 10 V/厘米 电泳凝胶,直至凝胶上样缓冲液中的染料距离凝胶末端 2-3 厘米。
  8. 根据凝胶估计探针大小的范围。大部分 DNA 斑迹应该在 300 - 3000 bp 范围内。当用于 CGH 分析时,较大的探针片段会使荧光强度降低。 

随着酶量的增加和孵育时间的延长,大小分布逐渐转移到更小的探针片段。要产生较小的探针片段,请使用以下条件,这些条件按片段大小递减的顺序列出:5 µL 酶混合物/2 小时孵育、5 µL 酶混合物/4 小时孵育、10 µL 酶混合物/2 小时孵育以及 10 µL 酶混合物/4 小时孵育。调节加入的无核酸酶水的量,以保持总反应体积为 50 µL。

 
 
 
 

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