Denaturing Gradient Gel Electrophoresis (DGGE)

Contact: Kristin Searcy

Reagent Preparation

1. Gel solutions of two different denaturing percentages need to be prepared.
   A broad denaturing gradient range such as 20-70% is typically used.  If you notice that most of the bands in your gel are congregated in one area, you may want to reduce the gradient range of your solutions so that the bands will be more separated.  The percent of acrylamide in the gel can also be varied depending on the size of the fragment being analyzed.   A 6% acrylamide gel is typically used for the separation of a 300-1000 bp fragment.
2. The following is a recipe for a 6% acrylamide gel. (For every additional 5ml of 40% Acrylamide/Bis added to the solution, the acrylamide gel percentage increases by 2%.)

40% Acrylamide/Bis                  15mL

50x TAE Buffer                          2 mL

Formamide                                  (40 x desired denaturing gradient) mL

Urea                                            (42 x desired denaturing gradient)  grams

Add dH₂O up to 100mL

(Note:  High percent denaturing solutions may need to be placed in a warm water bath to ensure complete dissolution.)

Transfer the solutions to two separate side arm flasks and degas the solutions for 10 minutes. Filter sterilize the solutions through a 0.45μm filter.  Store the solutions in a brown bottle at 4°C for up to one month.

Assembling Gel Sandwich

1. Clean the large and small glass plates with powder detergent and DI water .  Wear gloves and use your fingers to clean the plates; do not use a scrub brush.  Rinse the plates with DI water and prop them up vertically to air dry.
2. To assemble the gel sandwich lay the large rectangular plate on a clean surface . Place spacers along the right and left side of the glass plate.  The hole in the spacers should be near the top of the plate and the grooves near the hole should be facing down.
3. Place the short glass plate on top of the spacers and the long plate. Make sure the short plate is flush with the bottom edge of the long plate.
4. Place the sandwich clamps on the right and left side of the glass plates so that the arrows on the clamps are facing up and toward the plate.
5. Place the glass plates with spacers into the clamps and tighten the clamps enough to hold the plates in place by turning the knobs on the top clockwise.
6. Place the sandwich assembly upright in the alignment slot (the slot without the cams) of the casting stand with the short glass plate forward. Loosen the clamps and insert the alignment card.  To align the plates and spacers, push inward on both clamps while pushing down on the spacers with your thumbs, and then tighten both clamps just enough to hold the sandwich in place.
7. Remove the alignment card and remove the sandwich assembly from the casting stand. Check that the plates and spacers are flush at the bottom.  If they are not flush, realign the sandwich and spacers.  When a good seal is obtained, tighten the clamp screws.

Casting Denaturing Gradient Gels

1. Wet the gray sponge and place it in the front casting slot. Place the sandwich assembly on the sponge with the short plate facing you, and turn the handles of the camshaft so the cams lock the sandwich in place.
2. Attach tubing to two separate 30mL syringes.  With a separate piece of tubing place the Y-connector on one end of the tubing and attach a syringe needle on the other end.  Place, and secure with tape, the tip of needle between the two glass casting plates.
3. Make sure the cam wheel of the delivery system is in the vertical position by rotating it counterclockwise.
4. Transfer 16mL of the two denaturing solutions to two separate vials. When you are prepared to cast the gel, add 16μL TEMED and 160μL 10% ammonium persulfate to each vial and invert to mix.  (This step and the following 4 steps need to be completed in a timely fashion to prevent polymerization of the gel while casting.)
5. Using a 30mL syringe with the tubing attached, withdraw the LO denaturing gradient solution from the vial, and rid the syringe and tubing of any air bubbles. Place the syringe in the syringe sleeve on the LO density side of the delivery system by inserting the attachment screw on the syringe plunger in the lever groove.  Repeat this process with the HI density solution.
6. Connect the other end of the tubing from both syringes to the Y-fitting, which should be connected to the needle between the two glass plates.
7. Slowly rotate the cam wheel so that the gel solution is delivered to the gel sandwich over a 5 minute interval. Stop casting the gel when the solution is about 1 cm from the top of the small glass plate.
8. Immediately rinse the tubing with water so that the gel does not polymerize within the tubing.
9. Carefully insert the well comb into the gel, assuring that it is straight.  Allow the gel to polymerize for 60 minutes.

Gel Electrophoresis

1. Fill the DGGE electrophoresis tank to the line with 1x TAE (this requires approximately 7L of 1x TAE).
2. Place the lid on the apparatus making sure the stir rod is in the correct slot.
3. Preheat the system to 60°
4. After the DGGE gel has polymerized carefully remove the comb from the gel.  Slide and snap in the gel sandwich into the sandwich core using the knobs on the side of the gel sandwich.  Place the sandwich core with gel sandwich into the electrophoresis tank.
5. Load your amplified DNA sample with loading buffer into the wells of the gel using a flat tipped pipet tip.  (The DNA samples should be amplified using primers containing a GC clamp to prevent complete denaturing during electrophoresis.)
6. Run the gel at 60°C and 75 mV for 18 hours.
7. Remove the core sandwich from the electrophoresis tank, and remove the gel sandwich from the core sandwich.  Loosen the screws on the gel sandwich and remove the glass plates containing the gel.
8. Carefully separate the glass plates keeping the entire gel on one plate or the other.
9. Transfer the glass plate holding the gel to a staining box containing 1x SYBR Green.
10. Allow the gel to stain for at least 30 minutes.  Do not rock the gel during staining as this can cause the gel to tear.
11. Carefully transfer the gel from the staining box to the uv box to view the resulting bands. (The gel is very thin and fragile, so transfer the gel carefully to prevent tearing of the gel. An acetate sheet with holes punched in it can be used to support the gel when transferring it to the UV box.)