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DGGE Protocol – V1.0 (October 1999)
DGGE PCR Amplification
Per 50 µl rxn:
10X PCR Buffer* 5µl
50X dNTP 1
10X BSA 5
primer 341GCF 2 (20 pmol)
primer 907R 2 (20 pmol)
Taq pol 0.2 (1 U)
Template DNA 1-5
dH2O up to 50µl
Notes:
I would suggest using the Taq 2000 polymerase for environmental samples. Working tubes of primers should
be at 10 pmol/µl. The BSA is optional, I find it helps with environmental samples. Muyzer et. al. recommends
10-20 ng of template DNA.
These primers produce a ~600 bp product (including the gc clamp sequence).
Cycling Conditions
The MJ Cycler gives the best results. The Idaho is fast but the product yield is low.
MJ Cycler program is called “DGGE”
Initial denaturing: 3 minutes @ 95°C
Touchdown cycle: 30 sec. @ 95°C
30 sec @ 65°C, minus 1 degree per cycle for 10 cycles to 55°C
1 min. @ 72°C
Then 20 cycles of: 30 sec @ 95°C
30 sec @ 55°C
1 min @ 72°C
hold 4°C
After cycling, run 3-5 µl of product on agarose gel for quality check.
DGGE Reagents (from the BioRad Manual)
40% Acrylamide/Bis Solution (37.5:1)
Acrylamide 30g
Bis-acrylamide 2g
Add dH2O to 100 mls
Store in brown bottle at 4°C
Denaturing Acrylamide Solutions for a 6% Gel.
10% 20% 25% 50% 70%
40% Acrylamide/Bis 15ml 15ml 15ml 15ml 15ml
50X TAE buffer 2 2 2 2 2
Formamide (deionized) 4 8 10 20 28
Urea 4.2g 8.4g 10.5g 21g 29.4g
dH2O to 100ml to 100ml to 100ml to 100ml to 100ml
Store in a brown bottle at 4°C for about 1 month.
Gel Casting for Parallel DGGE (gradient and electrophoresis run in the same direction)
I run the gel overnight, at 70V for 16 or so hours. The buffer takes maybe an hour or so to heat up, and
pouring two gels also takes about an hour.
Heating up the buffer
Start up the buffer tank first since it has to heat up. Make up 8 liters of 0.5X TAE. Fill buffer tank with 7 L. Put
lid on, making sure stirring bar fits into support hole in tank. Plug it in, turn on power, heater, and set
temperature for 65°C. (I reuse TAE buffer for up to 3 runs).
1. Assemble gel sandwich – lay spacers on the long plate, grooved side down so that the holes are on the
top facing inward. Place short plate on top. Put the sandwich clamps on so that the long and short
plates fit into the notches in the clamps, and the arrows are in front. Tighten screws.
2. Place sandwich in the alignment slot, loosen clamps, put in alignment car d and tighten screws, making
sure everything is properly aligned.
3. The BioRad protocol doesn’t involve pouring a plug, but I’ve found the gel casting stand leaks and it’s
much easier to do a plug instead. Pouring plug for one gel: pipet 3 mls of the high denaturing
concentration acrylamide into plastic tube. Add 65 µl of APS and 5 µl of TEMED. Mix, run plug along
bottom of gel sandwich with pasteur pipet. Stand sandwich on glass plate until plug polymerizes.
4. While the plug polymerizes, prepare the denaturing solutions. The 16 x 16 cm gels take about 30 mls
of acrylamide. Half the volume will be the low denaturing solution and half will the high denaturing
solution. Set up two 50 ml plastic disposable tubes. Label on tube as ‘hi’, and the other as ‘lo’.
(Optional: Add 100-300 µl D-GENE dye solution to the tube marked ‘hi’). Pipet 15 mls of high
denaturing solution into the tube marked as high, and 15 mls of low denaturing into the other. (I usually
run 25-70% denaturing).
5. Make sure the camshafts on the casting stand are pointing up and are pulled out. Secure gels in
casting stand – place it on the grey foam, push down on sandwich clamp screw and turn the handles of
the camshaft so it points downwards. Do the same on the other side. Place comb in gel sandwich at
an angle.
6. Prepare the gradient delivery pump for use. Make sure the volume settings are correct (see manual)
for a top-filling gel. Set the pump for the 15 ml mark. Unwrap two 30 ml disposable syringes, marking
one as ‘hi’ and the other as ‘lo’. Place the syringe sleeves and plunger caps on the syringes. Make
sure the back of the sleeve is in the same plane as the level attachment screw. Connect the luer
fittings and the attached tubing to the syringes. Connect the tubing with the needle attached to the Y-
fitting. (Occasionally the Y-fitting has to be re-sealed with vacuum grease. Loosen plastic screws and
remove metal tubes. Put a small amount of grease around outside of tube at one end. Do not get this
inside of the tubes! Slide the tubes back in where the grease is and tighten screws). Make sure there
aren’t any water droplets in tubing.
7. To each tube of acrylamide, add 15µl TEMED, and 70-150 µl of APS. (Freshly made stock 40% stock
acrylamide will polymerize in about 30 min. with 70 µl of APS). Mix.
8. With the tubing connected to the syringe, pill all of the high-density solution into the syringe marked ‘hi’.
Air bubbles are ok at this time.
9. Remove all air bubbles by tapping syringe, and completely fill the tubing with gel solution (Very
Important!). Place the high-density syringe into the syringe holder on the pump (make sure it’s the
correct side). Attach the tubing to the Y-fitting. Fill the low-density syringe, remove bubbles, fill tubing
and place in pump. The sequence of insertion is very important. Make sure the plunger cap
attachment screws are seated in the groove.
10. Hold beveled edge of needle against the glass plate in the middle of the gel sandwich. Rotate the cam
wheel slowly and steadily to deliver gel solution. It should take about 5 minutes to fill the gel. If
bubbles form while pouring, knock on plate. Try to avoid bubbles up by the comb.
11. When finished, detach syringe tubing and rinse immediately to avoid gel polymerizing in the tubes.
12. Inspect the gel visually for gradient by color. If it is sagging a bit in the center from pouring, don’t worry,
this will slowly even out before it polymerizes.
13. Adjust comb carefully. With a pipettor, add a little TEMED to top of gel, making sure to get a bit on
each well wall, or else wells will be short.
14. Let polymerize for 30-40 minutes.
Running the DGGE Gel
1. Remove the comb, wash wells immediately with buffer.
2. Wet gasket on center core, snap gel sandwich into place. The short glass plate faces the core. If
running only one gel, put empty plates together with clamps and attach to other side to create wall for
upper buffer reservoir. Be very careful to put the sandwich clamps on the empty plates correctly, or
else plates will break.
3. Turn power off on lid, wait about 15 seconds for the heater to cool. Remove lid, rest on holder. Place
core into buffer tank. (It only goes in one way, with the BioRad writing on the right). Pour about 350 ml
0.5X TAE into upper buffer tank. Replace lid, plug back in, and turn power and pump on. Plug in
electrodes. Run at 200 V to pre-electrophorese until temperature is 62-65°C.
4. When buffer and gel are hot enough, turn off power, unplug electrodes and remove lid. Wash well
thoroughly. Add 5 µl of gel-loading buffer to each PCR product. Load gel using large gel-loading tips.
Wells will hold about 50 µl of sample. The amount of product to load varies with the sample. If it is
very diverse, load the whole amount. If you suspect low diversity, and you have a high product yield,
load 10-30 °C. Gently replace lid. Turn power and heater back on. Plug electrodes in. set
temperature for 60°C. Electrophorese for 5 minutes or so, then turn pump on. (This usually causes the
power supply to read ‘leak’, just keep turning it on until it stays on, or lower voltage and run for a few
minutes, the try again). Note: gel can be loaded by removing the top cover or the lid only, rather than
the whole lid assembly. This is a bit difficult, and it’s hard to wash the wells properly but it keeps the
buffer from cooling down.
5. Run gel for 16 hours at 70V and 60°C.
Staining
1. Place benchcote absorbent paper on bench, and place a piece of plastic wrap on top of it. Put 20 mls
of TAE buffer in a 50 ml centrifuge tube. Add 2-3 µl of SYBR Green to TAE, mix.
2. Turn all power off to system, let heater cool for 15 sec., then remove lid. Take out core. Unsnap gel
sandwich(s). Unscrew clamps, remove. Slide out spacers, and use them to separate the plates.
Remove top plate, and turn plate with gel attached over and transfer gel to plastic wrap. (Loosen one
end, and the gel should gently fall onto wrap). (Careful, the gels are fragile). Pour SYBR Green/TAE
solution onto gel. Cover with aluminum foil (SYBR Green is light sensitive). Stain for 15-30 minutes.
Destaining is not necessary.
3. Capturing gel image – remember to use the proper filter for the camera (It’s not the same as the one for
ethidium bromide).
Sequencing Bands from DGGE
1. Carefully cut out the band of interest from the gel.
2. Place in microfuge tube, spin to get acrylamide to bottom of tube. Sample can be frozen at this point
and stored.
3. Add 10-15 µl of sterile dH2O to tube, breaking up acrylamide slice with pipet tip.
4. Let DNA elute for 10-20 minutes at room temperature.
5. Use 1-2 µl of this as template in PCR reaction. Use primers 341F and 907R, with 55°C as the
annealing temperature. (Or use the “DGGE” cycle on the MJ).
6. Check quality of the PCR product on an agarose gel.
7. Use 1-2 µl of this as a template in a sequencing reaction, using the Licor labeled 341F and 907R
primers. I use 55°C for annealing. I also like to denature and cool the template before adding it to the
sequencing reaction mixture.
8. Following sequencing procedure as usual.
Plasmid Miniprep (Courtesy of Russ Karls and Vince Schultz)
1. Inoculate 4 mL LB (with appropriate antibiotics) with 1 colony. Incubate on shaker 37°C overnight.
2. Transfer 1-1.5 mL to Eppendorf tube, spin 10 sec. Pour off supernatant.
3. Re-suspend in 100 µL cold solution A. Put tube on ice.
4. Add 200 µL cold solution B. Mix by inversion. Leave on ice 5 min.
5. Add: 150 µL cold 7.5 M ammonium acetate
150 µL 24:1 chloroform: isoamyl alcohol
Mix by inversion.
6. Spin 5 min. in cold microfuge.
For cleaned-up minipreps:
7. Add supernatant (~400 µL) to 200 µL of 30% PEG 8000, 1.5 M NaCl. Mix. Leave on ice 15 min.
8. Spin 5 min. in cold microfuge. Remove all supernatant. Spin again to get all liquid (this is important
because PEG can inhibit many enzymatic reactions).
9. Re-suspend pellet in 50 µL DNA buffer (or sterile dH2O).
Solution A
Concentration For 10 mL
0.25 M Tris Cl (pH 7.8) 25 mM 1 mL
0.5 M EDTA (pH 8.0) 10 mM 0.2 mL
40% glucose 50 mM 0.25 mL
ddH20 8.5 mL
Solution B (make fresh weekly)
For 10 mL
10% SDS 1.0 mL
10 N NaOH 0.2 mL
ddH20 8.8 mL
