PROTOCOL FOR THE DGGE

EXPERIMENT & APPLICATION TO

MOLECULAR MICROBIAL ECOLOGY

Suk-Kyun Han
Question : sukkyun.han@ucr.edu
Reagent
1. 100% denaturing solution
z 40ml Formamide
z 42g Urea
z make to 100ml
z required to degas (If storage over 1 month)
2. 40% PAGE solution
3. 10% APS(ammonium persulfate) : make freshly
4. TEMED
5. DW

How to make Denaturing gel plate

When you run 30~50% & 40~60% denaturing gradient and 6% polyacrylamide gel
Stacking 0%
Composition High 60% (50%) Low 40% (30%)
denaturant
40% acrylamide sol. 1.80 1.80 0.6
50× TAE 0.24 0.24 0.08
100% denaturing sol. 7.20 (6.0) 4.80 (3.6) 0
DW 2.76 (3.96) 5.16 (6.36) 3.32
Total 12.00 12.00 4 ml

When you run 30~50% & 40~60% denaturing gradient and 8% polyacrylamide gel
Stacking 0%
Composition High 60% (50%) Low 40% (30%)
denaturant
40% acrylamide sol. 2.40 2.40 0.6
50× TAE 0.24 0.24 0.08
100% denaturing sol. 7.20 (6.0) 4.80 (3.6) 0
DW 2.16 (3.36) 4.56 (5.76) 3.32
Total 12.00 12.00 4 ml
 Stacking gel
(3.5ml capacity
when use 1mm
thick spacer)

Denaturing gel
(24ml capacity)

Materials
9 High (60%) denaturant concentration gel solution -Most of equipment is purchased in
9 Low (40%) denaturant concentration gel solution Biorad
9 10% ammonium persulfate (APS) -No need to degas when you use
9 TEMED solutions just prepared.
9 50 ml conical tubes
9 Gradient former set (30mL syringe, needle, tube,
gradient former)-Biorad
9 Gel plate set (glass plates, 1mm spacers, sandwich
clamps, plate assembler, alignment card) -We polish only one glass plate to make
the gel attached to only one side when
removing it.

9cm
-For the prevent the electric leakage
from the plate side, make sure assemble
without leaking and grease the silicone
15.5cm When no silicon grease is used on the
spacers, the DGGE bands will curve
down at the sides of the gel (weeping
bands instead of smiling researcher)
(Brinkhoff and van Hannen, 2001).
Using too much grease will deteriorate
Procedure the quality of the gel.
(1) clean plates with 95% EtOH and dry it.
(2) assemble the gel plate and spacers with sandwich clamps.
(3) place assembled plate onto the casting stand.
(4) fix the needle at the center of the plate as a picture
 -Step 6 is time sensitive only allowing 7-
10mins.

Note: if lab temperatures exceed 27°C

you may want to bring down the
concentration of APS and TEMED.
Elevated temperatures accelerate the
polymerization process

-Butanol layer will be helpful to make a

horizontal line and protect the oxygen.

(5) mix and prepare solution as above the table. -Stacking gel is help to make a same
(6) when you prepared high and low denaturing gel solutions, position when DNA is migrated on the
just add 100 ㎕ of 10% APS(use good quality of electrophoresis. All DNA molecules will
ammonium persulfate) and 10 ㎕ TEMED and then load be met the denaturing gel at the same
time.
the solution by gradient former immediately.
(7) Immediately after pouring, put 1 ml of water-saturated
butanol on top to obtain a straight surface. -Air bubble will be discarded between
(8) Let the gel polymerize for at least 60 minutes. gel and comb.
(9) After polymerization flush the space between the glass
plates by filling it 3 times with MilliQ to get rid of the -Do not leave the gel plate over 2
hours. It will be dried quickly when room
butanol. temperature is high.
(10) Carefully dry the space between the glass plates with
Whatman paper. -Must be used 2× loading dye
(11) Place the comb and fill up the gel chamber with a
mixture of 4ml stacking gel mixture, 5~10 µl TEMED and
50~80 µl APS using a pipette.
(12) Allow the stacking gel to polymerize for at least 30
minutes.
(13) Remove the comb and rinse the well with 1× TAE buffer.

Sample prepare
(1) amplify the PCR-DGGE DNA
(2) mix 20 ㎕ DNA and 20 ㎕ dye (2× loading dye)

Running
(1) Before the running, must turn on the Dcode system to
reach the temperature at 60℃ (at least 1 hour required).
(2) Gel plates are attached to the core and fill up the upper
reservoir with the TAE buffer.
(3) Use long-nose tips for loading the samples (mixed with
loading buffer).
(4) Load a marker along with the samples. (For determination of band positions and
comparability of gels we use 3 marker lanes: 1 in the middle and 2 at each side of the
gel).
(5) 2 gels can be placed on the core, when running 1 gel, a buffer dam is placed instead of
the second gel. This can be a piece of plastic with the same dimensions as the
assembled gel. But it can also be a gel chamber (the glass plate sandwich) in which a
1.5 mm thick plastic plate replaces the gel.
(6) Place the gel chamber in the cooling core. Loosen the
clamps a quarter counter clockwise to prevent breaking of
the sandwich clamps (due to heat-expansion).
(7) Turn on the pump and heat again
(8) Start power at 100V for 10 minutes and check the
amperage (about 20mA for one gel plate).
(9) After 10minutes, change the voltage to 50V and re-run for
800 minutes (or 70V for 500mins).
(10) Stop pump and power.*Xylene cyanol dye will be hung
on the end of gel.
(11) Remove gel from gel plate by removing the clamps and
Step 8. Not to be restricted. You may
one glass plate leaving the gel on the other. skip No. 8 in running step.
(12) Incubate gel on glass plate in 1 x TAE/EthidiumBromide;
15 min, shaking slowly. Make sure the gel is not sticking to
the glass plate while shaking.
(13) Remove the gel from the glass plate by sliding it off
carefully onto the UV illuminator. Wetting the UV
illuminator first will make sliding easier.
(14) Alternatively you can handle the gel by holding it at the
upper corners. Wet your gloves first. By gentle handling
(needs some practice) even bigger or thinner gels won’t
break.
(15) Make a photo picture

-The big image size is useful to analyze

the band profile, so make a picture
image as big as possible.
 Stacking gel
6% Polyacrylamide (3.5ml capacity
when use 1mm
thick spacer)

Denaturing gel
(24ml capacity)
40~60%

Stacking gel
8% Polyacrylamide (3.5ml capacity
when use 1mm
thick spacer)

Denaturing gel
(24ml capacity)
40~60%
DGGE pattern analysis by Gelcompar II or Bionumerics 5.1

The DGGE profiles were analysed using the software program BIONUMERICS 5.1
(Applied Maths BVBA). First, the DGGE banding patterns were converted to a binary
matrix. Using presence±absence data, a pairwise similarity of the banding patterns of the
different samples was calculated using the Dice coefficient Sd = 2j / (a+b), where j is the
number of bands common to both samples, a is the number of bands in sample A, and b is
the number of bands in sample B. This number is then multiplied by 100 to obtain the
percentage similarity. A value of 0 indicates that the samples are completely different,
whereas a value of 100 indicates that they are identical. Using these pairwise similarity
values, an UPGMA cluster analysis was conducted to determine whether the samples
revealed a non-random pattern and whether they clustered according to lake or rather
according to season irrespective of locality. Bootstrap values were calculated for each
dichotomy.

Principle Component Analysis

PCA was used to investigate the variation (presence / absence of bands) in the DGGE
banding patterns. PCA allows ordering of samples and taxa (i.e. bands) along axes
(principal components) on the basis of the banding patterns alone (Jongman et al., 1995).
The relationships between the ordination axes and measured environmental variables (both
abiotic and biotic) were analysed using correlation analyse (Spearman rank correlation).
The program was implemented using the CANOCO 4.0 for Windows software (ter Braak
and Smilauer, 1998). The results of the ordination analyses are visualized as biplots.
Pearson correlation [0.0%-100.0%]
BAC-DGGE(2) BAC-DGGE(2)

100
10

20

30

40

50

60

70

80

90
1B 9
1C 9
2B3B 9
1A 9
2A3A 9
5B 30
6A 16
2A3A 16
2B3B 16
2C3C 16
4B8B 16
4A8A 16
4C8C 16
1C 16
1B 16
4B8B 30
2C3C 9
Inflow 9
2C3C 30
1A 16
4A8A 9
4B8B 9
5A 9
5B 9
6B 9
7B 9
7A 9
6A 9
Inflow 16
Inflow 30
6C 30
7A 30
7B 30
7C 30
6A 30
6B 30
5C 30
7B 30
1A 30
1B 30
2B3B 30
2A3A 30
1C 30
4A8A 30
4C8C 30
5A 30
5A 16
5B 16
6B 16
5C 16
7A 16
1C 0
2C3C 0
1B 0
1A 0
2B3B 0
2A3A 0
5A 0
F-2C3C 0
7C 16
7B 16