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Preparation of Sequencing Gels

Preparation of Sequencing Gels
Contributor: The University of Maryland Baltimore County Applied Molecular Biology Program
1. Wash and rinse the sequencing gel plates.

2. Silanize the smaller plate by applying a small amount of Rain-Ex to the inside surface with a Kim Wipe.

3. Allow plate to air dry (see Hint #2).

4. Rinse the plate well with ddH2O and allow to dry.

5. Assemble the electrophoresis apparatus according to manufacturer's instructions.

6. Add Urea Gel Solution and allow gel to polymerize.

7. Add 1X TBE Buffer to the upper chamber.

8. Rinse unpolymerized acrylamide and urea from the comb-well area (see Hint #3).

9. Add 1X TBE Buffer to the upper chamber.

10. Pre-electrophoresis the gel at 35 to 50 watts for 30 to 60 min.

11. The gel should be warm to the touch.

12. Load preheated samples (preheat at 80°C for 5 min) to the warm gel.

13. Add 1 μl of 0.04% Bromophenol Blue to each lane that will contain sample.

14. Electrophoresis the gel at between 35 to 40 watts until Bromophenol Blue reaches near to the bottom of the glass plate.

15. Disassemble the plates carefully and make sure the gel sticks to the silanized plate.

16. Add Fixative Solution and let stand for greater than 15 min.

17. Drain excessive Fixative Solution from the gel and place 3MM Filter Paper on the gel.

18. Peel the gel off of the glass plate and onto the filter paper.

18. Wrap in Saran Wrap and gel dry.

19. Dry gel thoroughly on gel dryer for approximately 30 to 90 min at 80°C.

20. Remove Saran Wrap and expose to film for approximately 20 to 24 hrs.

Fixative Solution   5% (v/v) Glacial Acetic Acid
15% (v/v) Methanol
40% Acrylamide:Bis-Acrylamide   40% (19:1) Acrylamide:Bis-Acrylamide (CAUTION! see Hint #1)
0.04% (w/v) Bromophenol Blue
10% (w/v) Ammonium Persulfate
TBE Buffer   pH 8.0
89 mM Tris
89 mM Boric Acid
Urea Gel Solution   46 g of Urea (final concentration 8 M)
Stir gently and use immediately
Add 666 μl of 10% (w/v) Ammonium Persulfate
40 μl of TEMED
Add warm ddH2O to 100 ml
15 ml of 40% Acrylamide:Bis-Acrylamide (final concentration 6%)
10 ml of 10X TBE Buffer
BioReagents and Chemicals
Ammonium Persulfate
Boric Acid
Glacial Acetic Acid
Bromophenol Blue
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. The plate should have a visible thin film.

3. Make sure the well-comb area is completely free of unpolymerized material.


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