Electrophoresis - Polyacrylamide Gels
Jeff Lawrence
1. Pour and polymerize a polyacrylamide gel.
2. Remove clamps. Rinse with water. Remove comb. Rinse top of gel well.
3. Insert comb teeth down until teeth just touch the gel. Mount the plates in apparatus. Fill chambers with 0.5X TBE and prerun at low wattage for 10 min.
4. Force excess urea from the wells with a Pasteur pipet.
5. Denature samples and apply to gel. For 4-base restriction endonuclease gels, mark orientation with radioactive size standard loadings. For DNA sequencing gels, mark orientation by double 'G' lane loading. Load sequencing gels G A T C.
6. Force Urea from the wells between each loading. DNA samples do not need to be denatured more than once.
7. Run narrow gels at 35 W. Run wide gels at 80 W.
8. For sequencing gels run as follows:
Loading Xylene Cyanol Bromophenol Blue
Primer prox. Middle Just running off
Second 2/3 way down Off
Third Running off Off
9. Decant buffer from apparatus. Remove gel and lay flat.
10. Remove comb and side spacers. Pry apart plates with a spatula. If the gel is stuck to the top plate, resettle plates and invert.
11. Lift the upper plate off. If bubbles arise, wet gel with 10% Acetic Acid and work them out. Pat dry gently with a Kim-Wipe.
12. If 35S or 33P nuclides are used, soak gel 15 min in 10 % Acetic acid. Pat dry.
12. Cover gel with Whatman 3MM paper. Lift off gel stuck on paper. Cover with plastic wrap.
13. Dry in vacuum gel drier for 20 min at 80°. If soaked, dry 60 min.
14. If soaked, removed plastic wrap.
15. Expose to X-ray film.
Last Update: Thursday June 19 2014
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Eric Kofoid
eckofoid at ucdavis.edu