欢迎您, 登录 | 注册

首页 | English

X
加载中

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.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

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, 6767 views, 0 Q&A, How to cite
DOI: http://dx.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.

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.


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



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册