DGGE Protocol

PROTOCOL FOR THE DGGE

EXPERIMENT & APPLICATION TO

MOLECULAR MICROBIAL ECOLOGY

Suk-Kyun Han Question : [email protected]

Reagent 1. 100% denaturing solution

40ml Formamide 42g Urea make to 100ml 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 Composition High 60% (50%) Low 40% (30%) Stacking 0%

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 Composition High 60% (50%) Low 40% (30%) Stacking 0%

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

Materials

High (60%) denaturant concentration gel solution Low (40%) denaturant concentration gel solution 10% ammonium persulfate (APS) TEMED 50 ml conical tubes Gradient former set (30mL syringe, needle, tube, gradient former)-Biorad

Gel plate set (glass plates, 1mm spacers, sandwich clamps, plate assembler, alignment card)

Procedure

(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

Denaturing gel (24ml capacity)

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

-Most of equipment is purchased in Biorad -No need to degas when you use solutions just prepared. -We polish only one glass plate to make the gel attached to only one side when removing it.

-For the prevent the electric leakage from the plate side, make sure assemble without leaking and grease the silicone 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 the quality of the gel.

15.5cm

9cm

(5) mix and prepare solution as above the table. (6) when you prepared high and low denaturing gel solutions,

just add 100 ㎕ of 10% APS(use good quality of ammonium persulfate) and 10 ㎕ TEMED and then load the solution by gradient former immediately.

(7) Immediately after pouring, put 1 ml of water-saturated butanol on top to obtain a straight surface.

(8) Let the gel polymerize for at least 60 minutes. (9) After polymerization flush the space between the glass

plates by filling it 3 times with MilliQ to get rid of the butanol.

(10) Carefully dry the space between the glass plates with Whatman paper.

(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).

-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. -Stacking gel is help to make a same position when DNA is migrated on the electrophoresis. All DNA molecules will be met the denaturing gel at the same time. -Air bubble will be discarded between gel and comb. -Do not leave the gel plate over 2 hours. It will be dried quickly when room temperature is high. -Must be used 2× loading dye

(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

one glass plate leaving the gel on the other. (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

Step 8. Not to be restricted. You may skip No. 8 in running step.

-The big image size is useful to analyze the band profile, so make a picture image as big as possible.

Denaturing gel (24ml capacity) 40~60%

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

8% Polyacrylamide

Denaturing gel (24ml capacity) 40~60%

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

6% Polyacrylamide

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)

100

908070605040302010BAC-DGGE(2)

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1C

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1A

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4A8A

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Inflow

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5B

6B

7B

7A

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Inflow

Inflow

6C

7A

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7C

6A

6B

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7B

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1B

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1C

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1B

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7C

7B

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9

9

9

30

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30

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30

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30

16

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0

0

0

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0

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