By Tom Moss
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Extra resources for DNA'Protein Interactions: Principles and Protocols (Methods in Molecular Biology), Edition: 2nd
Remove the plastic wrap from the gel, cut out the marked bands with a scalpel or spatula, and cut them into small pieces. 1 mg/mL carrier DNA) and shake for several hours at room temperature. 4. Spin the tube for a few seconds in a microcentrifuge and transfer the liquid to a new tube. Avoid transferring gel pieces to the new tube. Add 200 µL of watersaturated neutralized phenol, shake 1 min, and centrifuge for 5 min at room temperature. Remove aqueous layer (upper), transfer it to a fresh Eppendorf tube, and add 30 µL of 3 M sodium acetate and 1 mL of ice-cold 100% ethanol.
40. Zhang, X. , Asiedu, C. , Supakar, P. , and Ehrlich, M. (1992) Increasing the activity of affinity-purified DNA-binding proteins by adding high concentrations of nonspecific proteins. Anal. Biochem. 201, 366–374. 41. , Fritsch, E. , and Maniatis, T. (1989) In Molecular Cloning, A Laboratory Manual , 2nd ed. ), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 42. , and Guérin, S. L. (1993) A simple apparatus for fast and inexpensive recovery of DNA from polyacrylamide gels. Biotechniques 14, 942–948.
This partially cut DNA is applied to a sequencing gel. If the DNA is detected by a unique terminal radioactive label, a DNA ladder is produced that is similar to that obtained by sequence analysis. Blanks within this regular ladder indicate the sites where the protein is bound. This footprint becomes more evident if a reference DNA is included that has been subjected to the same procedure but without previous protein binding. , the assay contains free DNA), the footprint will be masked by apparent cleavage within the protein-binding site.