MOLECULAR BIOLOGY TOOLS (MBT) WORKSHOP

DGGE analysis

Braga, University of Minho 23-24 June 2013

Supported by:

CONTENTS:

1. DNA extraction 2. Polymerase chain reaction (PCR) 3. Assembling the DGGE plate cassette 4. Casting the denaturing gradient gels 5. Running the gel 6. Staining the gel

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1. DNA extraction
There are several commercial kits for DNA extraction. In this course, total DNA will be extracted from approximately 500 l of sample by using Fast DNA Spin kit for soil (QBio gene), according to the manufacturers protocol. The extracted DNA is maintained at -20C or at -80C for longer periods.

2. Polymerase chain reaction (PCR)

10-fold serial dilutions from extracted DNA are made and tested in the PCR to find the concentration of template DNA that gives a good specific product. Table 1 shows the volume of each reagent used in one polymerase chain reaction. A Taq DNA Polymerase kit (Life technologies) is used, which provides Taq DNA Polymerase itself in addition to PCR reaction buffer and MgCl2.
Table 1 Reagents and respective volumes used in one polymerase chain reaction.
10 PCR reaction buffer (without MgCl2) MgCl2 dNTPs (10 mM each) Forward primer (10 M) Reverse primer (10 M) PCR water Taq DNA polymerase (5 unities/ l) Total volume 5 l 3 l 1 l 1 l 1 l 37.75 l 0.25 l 49 l

A volume of 1 l of DNA template (resulted directly from the DNA extraction or from the dilutions) is added to each PCR reaction tube containing 49 l of PCR mix (Table 1). The tubes are inserted into the thermocycler and the appropriate PCR program is selected (according to the primers selected).

MBT Workshop, 23-24 June University of Minho, Braga, Portugal

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The primer set U968GC-f/L140-r (Nbel et al 1996) is often used to assess bacteria diversity by DGGE, with the following PCR program: 95C 95C 56C 72C 72C 4C 2min 30s 40s 1min 5min 1x 35x 1x

The size of the obtained PCR products is checked by comparison with appropriate size and mass standard by electrophoresis on an 1% (w/v) agarose gel. SYBRSafe staining is applied. A negative control is also loaded in the agarose gel, which consisted of the PCR products obtained from a 49 l PCR mix without DNA template addition. Gels run at a constant voltage of 100 V in an agarose gel electrophoresis system (Mupid-EXU). Nucleic acids are detected in an UV transilluminator (BioRad). Reagents and solutions preparation are described below.

PCR Water: Filter Milli-Q water with sterile filters (0.2 m) and autoclave (121C, 15 min). Distribute aliquots of 1 ml into sterile eppendorfs in a PCR/UV work station. Store at -20C.

1% (w/v) agarose gel: 75 ml TAE 0.5x 0.75 g agarose 7.5 l SYBR Safe

Dissolve agarose in microwave oven. Add the SYBR Safe and pour into the gel tray.
MBT Workshop, 23-24 June University of Minho, Braga, Portugal

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50x TAE buffer (pH 8.3): 242 g Tris base 57.1 ml acetic acid, glacial 100 ml EDTA 0.5 M (pH 8.0)

Adjust to 1 L with dH2O. Autoclave for 20 minutes. Store at room temperature.

DNA loading buffer: 46.8 ml glycerol 80% 76.7 ml dH2O 1.5 ml EDTA 0.5 M 0.15 g bromophenol blue 0.15 g xylene cyanol FF

dNTP solution: 100 l each dNTP (from 100 mM dNTP set) 600 l PCR water

Distribute 100 l to 10 sterile eppendorfs.

MBT Workshop, 23-24 June University of Minho, Braga, Portugal

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3. Assembling the DGGE plate cassette

Clean one large and a smaller glass plate with soap, dry them and clean them again with 96% ethanol. Cut the gelbond (Cambrex Bio Science) to the size of the largest glass plate. Add some water to the surface of the large glass plate Place the gelbond hydrophobic side down on this glass plate (you can easily check this by adding a drop of water on the gelbond, it will roll of easily). Fix the gelbond, without removing the paper sheet, with a roller, then take of the paper sheet. Dry the gelbond carefully with some tissue. Clean a set of spacers with 96% ethanol, and place them on the gelbond. Place the smaller glass plate on top. Add the clamps to the sides of the sandwich, and place in the sandwich-holder. For proper distance between the spacers slide the plastic card between the glass plates. Press the spacers down and fasten the screws on the clamps. Place the sandwich on top of the rubber gasket and press down the handles (dont use to much force)

MBT Workshop, 23-24 June University of Minho, Braga, Portugal

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4. Casting the denaturing gradient gels

Prepare the gel solutions (HIGH, LOW, plug and stacking gel) in 50 ml tubes, on ice, as described on Table 2 (the APS solution is added just before starting pouring the gel). For bacteria amplicons analysis, a 30-60 % gradient is usually used. Prepare 1 ml of plug gel solution (1,5 ml 0 % solution + 4,5 ul TEMED + 15 ul APS) and pour it at the bottom of the DGGE plate cassette (between glass plates) prior to make the main resolving gradient gels. It will prevent leakage from the bottom of the plates. For casting gradient gels, a gradient former, a magnetic stirrer and a peristaltic pump (BioRad) is used. Rinse the gradient maker and tubes with demi-water, switch on the pump at running speed (20 ml/min) and drain the system. Close the screw between the compartments of the gradient maker. Dry the compartments with a tissue. Add 10% APS to the HIGH and LOW solutions and pour it in the right and left compartment, respectively. Start the stirrer. Open the screw and immediately start the pump at 4 ml/min. Place the needle between the glass plates and secure the tube. Remove the needle when the gel is poured, switch off the pump and transfer to the erlenmeyer flask. Rinse the compartments with demi-water, switch on the pump and drain the system.

MBT Workshop, 23-24 June University of Minho, Braga, Portugal

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Table 2 Denaturing gradient gel electrophoresis mixing table.

Gradient Comp. % 0 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 0% Solution ml 9,0 9,8 9,6 9,4 9,3 9,1 8,9 8,8 8,6 8,5 8,3 8,1 8,0 7,8 7,6 7,5 7,3 7,2 7,0 6,8 6,7 6,5 6,3 6,2 6,0 5,9 5,7 5,5 5,4 5,2 5,0 4,9 4,7 4,6 4,4 4,2 4,1 3,9 3,7 3,6 3,4 3,3 3,1 2,9 2,8 2,6 2,4 2,3 2,1 2,0 1,8 1,6 1,5 1,3 1,1 1,0 0,8 0,6 0,5 0,3 0,2 0,0 80% Solution ml 0,0 3,3 3,4 3,6 3,7 3,9 4,1 4,2 4,4 4,6 4,7 4,9 5,0 5,2 5,4 5,5 5,7 5,9 6,0 6,2 6,3 6,5 6,7 6,8 7,0 7,2 7,3 7,5 7,6 7,8 8,0 8,1 8,3 8,5 8,6 8,8 8,9 9,1 9,3 9,4 9,6 9,8 9,9 10,1 10,2 10,4 10,6 10,7 10,9 11,1 11,2 11,4 11,5 11,7 11,9 12,0 12,2 12,4 12,5 12,7 12,8 13,0 Volume ml 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 TEMED l 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 10% APS l 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50

MBT Workshop, 23-24 June University of Minho, Braga, Portugal

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Add the 10% APS to the stacking gel (0% Gradient Comp.). Close the screw between the compartments and add the stacking gel to the right compartment. Start the pump and set at running speed (20 ml/min) until the solution has reached the needle and a few drops have dripped in the erlenmeyer flask. Stop the pump. Place the needle between the glass plates and start the pump set at 1 ml/min. Slowly, increase to 4ml/min. When the stacking gel is poured place the comb that will form the slots carefully in the stacking gel. Avoid air-bubbles since the will appear as dents in all your bands. Leave the gel for 1 hr.

5. Running the gel

Add fresh buffer to the buffer tank and fill up to Fill Switch on the Dcode at least 90 minutes before electrophoresis, so that the buffer can heat up to 60oC After one hour of gel polymerisation remove the comb carefully. Rinse off non-polymerized gel in and above the slots with demi-water and click the sandwich in the sandwich-holder. (There should always be a sandwich at the other side to get a closed upper buffer compartment). Switch off the Dcode, and take off the lid. Take this super-sandwich and let it slide into the buffer tank. Switch on the Dcode until the upper buffer compartment is filled with buffer.

MBT Workshop, 23-24 June University of Minho, Braga, Portugal

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Switch off the Dcode and take of the lid. Rinse all slots with syringe and needle filled with buffer. Add your samples (remember you have to load mirror-wise). Switch on the Dcode after returning the lid. Switch on the power supply for 10 min. at 200V and lower to 85V for 16 hrs.

6. Staining the gel

DGGE gels can be stained by different methods. For instance, it can be stained with silver according to the following (Sanguinetty et al. (1994)): -Placed the gel in a stainless steel box. -Add 200 ml 1xCairns fixation solution and rock the box for 3 min, remove the solution and store for later application. -Add 200 ml silver staining solution (caution: use gloves) to the box and rock 10 min. -Discard the solution (chemical waste!). -Rinse the box with demi-water (chemical waste!). -Add fresh demi-water and rock for 2 min. -Discard water (chemical waste!). -Rinse the gel and gel bond with demi-water. -Place the gel in a stainless steel developer tray. Add a small part of developer solution and rock a little, discard this part and replace by the remaining developer solution. -Rock until the gel has developed well.

MBT Workshop, 23-24 June University of Minho, Braga, Portugal

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-Discard the developer solution. -Add the previously used 200 ml 1x Cairns' solution to the box and rock for 5 min. -Discard the fixation solution. -Add some demi-water and rock for 2 min. -Replace the demi-water with Cairns' preservation solution and rock for 7 min. -Place the gel on a glass plate (gelbond faced down). -Pre-wet cellophane foil in the preservation solution and put it on the gel (avoid air bubbles, press the edges of the gel to the glass plate) Dry the gel overnight at 60C

Stock solutions 80 % denaturant, 8% PAG: 200 ml 320 ml 10 ml 20 ml 337,3 g 40% acryl/bisacrylamide 37.5:1 formamide 50x T.A.E. buffer glycerol (only when silver staining is performed) urea

Carefully heat to handwarm, add stirrer and dissolve. Adjust to final volume of 1 L with demi-water. Store in the dark at room temperature. 0% denaturant, 8% PAG: 200 ml 10 ml 20 ml 40% acryl/bisacrylamide 37.5:1 50x T.A.E. buffer glycerol (only when silver staining is performed)

Adjust to final volume of 1 L with demi-water. Store in the dark at room temperature.
MBT Workshop, 23-24 June University of Minho, Braga, Portugal

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10% Ammonium persulfate (APS): Store at -20 C in 500 l aliquots. Cairns 8x fixation solution: 200 ml 96% ethanol 10 ml acetic acid 40 ml dH2O 1g ammonium persulfate 10 ml dH2O

Silver staining solution: 0.4 g AgNO3 200 ml of 1x Cairns fixing solution

Developer solution: a spatula tip of NaBH4 (approx. 10 mg) 250 ml 1.5% NaOH solution 750 l formaldehyde

Cairns preservation solution: 250 ml 96% ethanol 100 ml glycerol 650 ml dH2O

MBT Workshop, 23-24 June University of Minho, Braga, Portugal

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References Nbel U, Engelen B, elske A, naidr J, ieshuber A, mann RI, udwig W, ackhaus H (1996) Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. Journal of Bacteriology 178:5636-5643. Sanguinetti CJ, Dias Neto E, Simpson AJ (1994) Rapid silver staining and recovery of PCR products separated on polyacrylamide gels. Biotechniques 17:914-921.

MBT Workshop, 23-24 June University of Minho, Braga, Portugal