Protocol

Preparation of Denaturing Polyacrylamide Gels

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

INTRODUCTION

In 1978, Fred Sanger and Alan Coulson devised a method to pour and run thin polyacrylamide gels, which are now used ubiquitously to resolve the products of DNA sequencing reactions.

MATERIALS

10x TBE electrophoresis buffer

TBE is used at a working strength of 1x (89 mM Tris-borate, 2 mM EDTA) for polyacrylamide gel electrophoresis.

Acrylamide solution (45% w/v)

Ammonium persulfate (1.6% w/v) in H₂O

Deionized H₂O

Detergent, household dishwashing

Ethanol

Optional, please see Step 5.

KOH/Methanol solution

Silanizing fluid

The traditional silanizing fluids (e.g., Sigmacote from Sigma and Repelcote from BDH Inc.) contain dichlorodimethylsilane, which is toxic, volatile, and highly flammable. In recent years, nontoxic alternatives have become available, including Gel Slick (FMC Bioproducts), RainX (Unelko, Scottsdale Arizona), and Acrylease (Stratagene).

TEMED (N,N,N',N'-tetramethylethylenediamine)

Electrophoresis-grade TEMED is sold by many manufacturers including Sigma and Bio-Rad. TEMED is hygroscopic and should be stored in a tightly sealed bottle at 4°C.

Urea, solid

METHOD

1. If necessary, remove old silanizing reagent from plates by swabbing them with KOH/methanol solution.

IMPORTANT
To prevent contamination of the glass surfaces by skin oils, wear talc-free gloves at all times and handle the plates by their edges.

2. Wash the plates and spacers in a warm, dilute solution of dishwashing liquid and then rinse them thoroughly in tap water, followed by deionized H₂O. Rinse the plates with absolute ethanol to prevent water spots, and allow them time to dry.

3. Treat the inner surface of the smaller or notched plate with silanizing solution. Lay the plate, inner surface uppermost, on a pad of paper towels in a chemical fume hood, and pour or spray a small quantity of silanizing fluid onto the surface of the plate. Wipe the fluid over the entire surface with a pad of Kimwipes and then allow the fluid to dry in the air (1-2 minutes). Rinse the plate first with deionized H₂O and then with ethanol, and allow the plate time to dry.

4. Lay the larger (or unnotched) glass plate (clean side up) on an empty test tube rack on the bench and arrange the spacers in place along each side of the glass plate so they are flush with the bottom of the plate.

5. Center the shorter (notched) plate, siliconized side down, on top of larger (unnotched) plate. Make sure that the spacers remain in position at the very edges of the two plates.

6. Clamp the plates together on one side with two or three large (5-cm length) bulldog binder clips. Bind the entire length of the other side and the bottom of the plates with gel-sealing tape to make a watertight seal.

7. Remove the bulldog clips and seal this side of the gel plates with gel-sealing tape.

8. Place the flat side of the sharkstooth comb into the open end of the gel mold so that it fits snugly. Remove the comb and lay the empty gel mold on the test-tube rack.

9. Cover the working area of the bench with plastic-backed protective paper.

10. In a 250-ml side-arm flask, prepare a sequencing gel solution containing the desired concentration of acrylamide as specified in the table below. The volumes given in the table are sufficient for a single 40 x 40-cm sequencing gel and can be proportionally adjusted to accommodate smaller or larger gels.

IMPORTANT
The preparation of the gel must be completed without interruption from this point onward.

View larger version (2K): [in this window] [in a new window]

11. Combine all of the reagents and then heat the solution in a 55'C water bath for 3 minutes to help dissolution of the urea.

12. Remove the solution from the water bath and allow it to cool at room temperature for 15 minutes. Swirl the mixture from time to time.

13. Attach the side-arm flask to a vacuum line and de-gas the solution.

14. Transfer the solution to a 250-ml glass beaker. Add 3.3 ml of freshly prepared 1.6% ammonium persulfate and swirl the gel solution gently to mix the reagents.

15. Add 50 µl of TEMED to the gel solution and swirl the solution gently to mix the reagents. Pour the gel solution into the mold directly from the beaker in which it has been prepared. Alternatively, draw approx. 40 ml of the solution into a 60-cc hypodermic syringe. Do not suck air bubbles into the syringe!

Compared with polyacrylamide gels used to resolve proteins, a massive amount of TEMED is used to cast sequencing gels. The large amount of TEMED ensures that polymerization will occur rapidly and uniformly throughout the large surface area of the gel. Because the rate of polymerization is temperature-dependent, cooling the gel solution allows more time for casting the gel. Experienced gel pourers can often cast two or more 40 x 40-cm gels from a single gel solution by judicious precooling.
Work as quickly as possible from here onward because the gel solution will polymerize rapidly. Polymerization can be appreciably slowed by putting the gel solution on ice, a boon for inexperienced gel pourers!

16. Allow a thin stream of gel solution to flow from the beaker or syringe into the top corner of the gel mold while holding the mold at an approx. 45' angle to the horizontal.

17. Lay the mold down on the test-tube rack (please see Figure 12-9 in the print version of the manual).

18. Immediately insert the flat side of a sharkstooth comb approx. 0.5 cm into the gel solution. Insert both ends of the comb into the fluid to an equal depth so that the flat surface is level when the gel is standing in a vertical position.

19. Clamp the comb into position using bulldog binder clips. Use the remaining acrylamide/urea solution in the hypodermic syringe/pipette to form a bead of acrylamide across the top of the gel. Allow the gel to polymerize for at least 15 minutes at room temperature.

20. Wash out the 60-cc syringe so that it does not become clogged with polymerized acrylamide.

21. After 15 minutes of polymerization, examine the gel for the presence of a Schlieren line just underneath the flat surface of the comb. This is a sign that polymerization is occurring satisfactorily. When polymerization is complete (approx. 1 hour after the gel was poured), remove the bulldog clips.

22. The gel can be used immediately (please see Loading and Running DNA Sequencing Gels) or stored for up to 24 hours at room temperature or 48 hours at 4°C. To prevent dehydration during storage, leave the tape in place and surround the top of the gel with paper towels dampened with 1x TBE. Cover the paper towels with Saran Wrap. Do not remove the comb at this stage.

REFERENCES

1. Sanger, F. and Coulson, A.R. 1978. The use of thin acrylamide gels for DNA sequencing. FEBS Lett. 87: 107–110.[Medline]