Welcome guest, Login | Register

Home

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

Molecular Biology > DNA > DNA labeling
Author: 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, 6749 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



Reproducibility Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Q&A and Troubleshooting:

  • Add Photo
  • Add Video

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register