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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, 7030 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 RPN 1606 (Amersham)

2.     Amersham Microspin G-50吸附柱27-5330-01(Amersham)

3.     dCTP-apha-32P 3000 Ci/mmol (PerkinElmer)

 

操作步骤:

1.     标记反应:

1)   在管中加入溶于28 ul TE 或者水中的25 ng 模板。加入试剂盒中的5 ul引物。

2)   100oC变性5分钟。

3)   点甩离心管。

4)   室温下加入10 ul 标记缓冲液,5 ul dCTP2 ul酶。

5)   混匀,37 温浴10分钟。

6)   加入5 ul  0.2 M EDTA终止反应。

2.     纯化探针:

1)   使用柱子时先涡旋以重悬柱子的基质。将盖子松开? 放入一个1.5 ml 螺口管 735 g离心1分钟. 离心的同时计时。

2)   将柱子放入一个新的1.5 ml螺口管,往柱子中心缓慢加入50 ul 样品。不要丢弃收集管。不要让样品从收集管边缘流出。

3)   735 g离心2分钟。

4)   纯化的样品在管底。

5)   加热然后冰上放置帮助杂交

6)   用闪烁计数器检测1ul探针样品。

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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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