Radioactive DNA bands separated by acrylamide gel electrophoresis can be detected by radioautography. Polyacrylamide analytical gels containing radioactive DNA are usually fixed and dried prior to radioautography. However, if radioactive DNA bands are to be recovered from the gel, they should not be fixed and dried. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Radioautography detection of DNA in polyacrylamide gels
Principle
Radioactive DNA bands separated by acrylamide gel electrophoresis can be detected by radioautography. Polyacrylamide analytical gels containing radioactive DNA are usually fixed and dried prior to radioautography. However, if radioactive DNA bands are to be recovered from the gel, they should not be fixed and dried.
Materials and Instruments
Acetic acid Move I. Materials For more product details, please visit Aladdin Scientific website.
Polyacrylamide gel Commercially available gel dryer Plastic sieve Radioactive ink or chemiluminescent standard reference Filter paper
1. Buffers and solutions
Acetic acid
2. Gels
Polyacrylamide gel
3. Specialized equipment
Commercially available gel dryer (optional or not)
Plastic sieve (1 cm mesh size, available at garden and hardware stores) (optional or not)
Radioactive ink or chemiluminescent standard reference material
Whatman 3 MM filter paper
II. Methods
1. Immerse the gel and its attached glass plate in 7% acetic acid for 5 min. carefully pick up the glass plate from the fixative to remove the gel.
2. Briefly rinse the gel in deionized water and dip a wad of Kimwipes paper into the gel to remove excess liquid from the gel surface.
3. (May or may not be done) Place the gel on Whatman 3 MM paper and dry in a commercially available gel dryer.
4. Wrap the gel and supporting glass plate in Saran Wrap film and remove any air bubbles or wrinkles in the Saran Wrap film with the wide end of a comb or folded Kimwipe paper.
5. Apply small adhesive labels labeled with radioactive ink or chemiluminescent standard reference to the surface of the Saran Wmp membrane to align the gel with the film. Cover the radioactive ink label with clear tape to avoid contaminating the film cartridge and sensitizing screen.
6. Invert the gel and expose the x-ray film (e.g., Kodak XAR-5 or equivalent) as follows:
(1) In a darkroom, tape the sealed gel to a sheet of X-ray film cut to the size of a glass plate.
(2) Wrap the gel and film in opaque aluminum foil.
(3) Expose the gel at room temperature or -70°C for an appropriate period of time, with or without a sensitizing screen.
(4) Develop, fix, and dry the x-ray film according to the manufacturer's recommendations.