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To probe for a specific mRNA species by Northern blot, RNA from the agarose/formaldehyde gel needs to be transferred to a nylon membrane. RNA is detected by hybridization using a labeled probe. The probe is a DNA or RNA molecule that is chemically or radioactively labeled. In this protocol synthesis and preparation of a [32P]-dCTP-labeled probe is described.

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32P Radioactive Probe Synthesis and Preparation
磷-32放射性探针的合成与制备

分子生物学 > DNA > DNA 标记
作者: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
Vol 1, Iss 1, 1/5/2012, 7476 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.7

[Abstract] To probe for a specific mRNA species by Northern blot, RNA from the agarose/formaldehyde gel needs to be transferred to a nylon membrane. RNA is detected by hybridization using a labeled probe. The probe is a DNA or RNA molecule that is chemically or radioactively labeled. In this protocol synthesis and preparation of a [32P]-dCTP-labeled probe is described.

[Abstract] 通过Northern 印记来探测某一特定mRNA,首先将琼脂/甲醛树脂凝胶上的RNA转移至硝酸纤维素膜上,用标记的探针对RNA进行检测。探针是用化学物质或者同位素标记的DNA或者RNA,作者使用的是[32P]-dCTP标记探针

Materials and Reagents

  1. Amersham Megaprime DNA labeling system (GE Healthcare Life Sciences, catalog number: RPN 1606 )
  2. Amersham Microspin G-50 Columns (GE Healthcare Life Sciences, catalog number: 27-5330-01 )
  3. dCTP-apha-32P 3000 Ci/mmol (PerkinElmer)
  4. TE buffer
  5. EDTA

Equipment

  1. Scintillation counter
  2. Bench-top centrifuge
  3. Heat block
  4. 37 °C incubator

Procedure

  1. Labeling reaction
    1. Place 25 ng of template in 28 μl TE or water in tube. Add 5 μl of primer solution from kit.
    2. Denature by incubating at 100 °C for 5 min.
    3. Spin tube briefly to bring contents of tube to bottom.
    4. At room temperature, add 10 μl of labeling buffer, 5 μl of dCTP, and 2 μl of enzyme.
    5. Mix and incubate at 37 °C for 10 min.
    6. Stop reaction by addition of 5 μl of 0.2 M EDTA.
      .
  2. Probe purification
    1. Prepare column by vortexing to resuspend matrix. Loosen cap ¼ turn and insert into 1.5 ml screw cap tube.
    2. Spin 1 min at 735 x g. Start timer and centrifuge simultaneously.
    3. Place column in a new 1.5 ml screw cap tube and slowly apply 50 μl of sample to center of column matrix.
    4. Do not disturb the column bed. Do not let sample flow around the sides of the bed.
    5. Spin column for 2 min at 735 x g.
    6. Purified sample is now at the bottom of the tube.
    7. Heat to 100 °C and chill on ice prior to adding to hybridization.
    8. Count 1 μl of probe in scintillation counter.

Acknowledgments

This protocol has been modified and adapted in the Espenshade Lab, Johns Hopkins School of Medicine. Funding to support different projects that have used this protocol has come from NIH – National Heart, Lung, and Blood Institute, National Institute of Allergy and Infectious Diseases, the Pancreatic Cancer Action Network, and the American Heart Association.

References

  1. Stewart, E. V., Nwosu, C. C., Tong, Z., Roguev, A., Cummins, T. D., Kim, D. U., Hayles, J., Park, H. O., Hoe, K. L., Powell, D. W., Krogan, N. J. and Espenshade, P. J. (2011). Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex. Mol Cell 42(2): 160-171.

材料和试剂

  1. Amersham Megaprime DNA标记系统(GE Healthcare Life Sciences,目录号:RPN 1606)
  2. Amersham Microspin G-50柱(GE Healthcare Life Sciences,目录号:27-5330-01)
  3. dCTP-apha- 32 32 P 3000 Ci/mmol(PerkinElmer)
  4. TE缓冲液
  5. EDTA

设备

  1. 闪烁计数器
  2. 台式离心机
  3. 热块
  4. 37℃培养箱

程序

  1. 标记反应
    1. 将25ng模板置于28μlTE或管中的水中。 从试剂盒中加入5μl底漆溶液。
    2. 通过在100℃孵育5分钟来变性
    3. 短暂旋转管,将管内容物放到底部
    4. 在室温下,加入10μl标记缓冲液,5μldCTP和2μl酶
    5. 混合并在37℃孵育10分钟。
    6. 通过加入5μl0.2M EDTA停止反应 。
  2. 探针纯化
    1. 通过涡旋制备柱以重悬基质。 松开盖¼转并插入1.5毫升螺旋盖管。
    2. 在735摄氏度旋转1分钟x g 。 同时启动计时器和离心机。
    3. 将柱放置在新的1.5 ml螺旋盖管中,并缓慢地将50μl样品加到柱基质的中心。
    4. 不要打扰柱床。 不要让样品流到床边。
    5. 在735分钟时旋转柱2分钟。。
    6. 纯化的样品现在在管的底部
    7. 加热至100℃并在加入杂交之前在冰上冷却。
    8. 在闪烁计数器中计数1μl探针。

致谢

该协议已经在约翰霍普金斯医学院的Espenshade实验室中修改和改编。 资助支持不同的项目,使用这个协议来自NIH - 国家心脏,肺和血液研究所,国家过敏和传染病研究所,胰腺癌行动网络和美国心脏协会。

参考文献

  1. Stewart,EV,Nwosu,CC,Tong,Z.,Roguev,A.,Cummins,TD,Kim,DU,Hayles,J.,Park,HO,Hoe,KL,Powell,DW,Krogan,NJ和Espenshade, (2011)。 酵母SREBP裂解激活需要Golgi Dsc E3连接酶复合物。细胞 42(2):160-171。
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How to cite this protocol: Tong, Z. (2011). 32P Radioactive Probe Synthesis and Preparation. Bio-protocol 1(1): e7. DOI: 10.21769/BioProtoc.7; Full Text



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