From Sample To DGGE Page 1 of 8
DNA isolation
For filters:
Place filters on piece of clean aluminium foil or petri dish.
Cut the filters with a flame-sterilized scissor, in ca. 0.3 cm wide strips.
Insert into the tubes at step.
Isolation according to protocols
MoBio Kit
Please wear gloves at all times
1. To the PowerBead Tubes provided, add 0.25 gm. of soil sample.
2. Gently vortex to mix.
3. Check Solution C1. If Solution C1 is precipitated, heat solution to 60°C until dissolved
before use.
4. Add 60µl of Solution C1 and invert several times or vortex briefly.
5. Secure PowerBead Tubes, place them in the Bead-Bead. Process at speed 4.0, for 40 seconds.
6. Make sure the PowerBead Tubes rotate freely in your centrifuge without rubbing. Centrifuge
tubes at 10,000 x g for 30 seconds at room temperature. CAUTION: Be sure not to exceed
10,000 x g or tubes may break.
7. Transfer the supernatant to a clean 2 ml Collection Tube (provided). Note: Expect between
400 to 500µl of supernatant. Supernatant may still contain some soil particles.
8. Add 250µl of Solution C2 and vortex for 5 seconds. Incubate at 4°C for 5 minutes.
9. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.
10. Avoiding the pellet, transfer up to, but no more than, 600µl of supernatant to a clean 2 ml
Collection Tube (provided).
11. Add 200µl of Solution C3 and vortex briefly. Incubate at 4°C for 5 minutes.
12. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.
13. Avoiding the pellet, transfer up to, but no more than, 750µl of supernatant into a clean 2 ml
Collection Tube (provided).
14. Add 1200µl of Solution C4 to the supernatant and vortex for 5 seconds.
15. Load approximately 675µl onto a Spin Filter and centrifuge at 10,000 x g for 1 minute at
room temperature. Discard the flow through and add an additional 675µl of supernatant to
the Spin Filter and centrifuge at 10,000 x g for 1 minute at room temperature. Load the
remaining supernatant onto the Spin Filter and centrifuge at 10,000 x g for 1 minute at room
temperature. Note: A total of three loads for each sample processed are required.
16. Add 500µl of Solution C5 and centrifuge at room temperature for 30 seconds at 10,000 x g.
17. Discard the flow through.
18. Centrifuge again at room temperature for 1 minute at 10,000 x g.
19. Carefully place Spin Filter in a clean 2 ml Collection Tube (provided). Avoid splashing any
Solution C5 onto the Spin Filter.
20. Add 100µl of Solution C6 to the center of the white filter membrane. Alternatively, sterile
DNA-Free PCR Grade Water may be used for elution from the silica Spin Filter membrane at
this step (MO BIO Catalog No. 17000-10).
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From Sample To DGGE Page 2 of 8
21. Centrifuge at room temperature for 30 seconds at 10,000 x g.
22. Discard the Spin Filter. The DNA in the tube is now ready for any downstream application.
No further steps are required. We recommend storing DNA frozen (-20° to -80°C).
DNA isolation using Biozyme FastPrep kit
Reagents and tubes are from the Biozyme FastPrep kit.
1. Add up to 500mg of soil or 0.5 ml liquid into Lysing Matrix E Tube (for filters, just insert
the pieces, do not add solution).
Due to the vigorous motion of the FastPrep® Instrument, a significant pressure buildup is observed in the
tube. The sample and the Lysing Matrix should not exceed more tha 7/8 of the tube in volume. Leaving space
in the tube also improves chances for better homogenization. [See Note 1,]
Add 900µl Sodium Phosphate Buffer and 120µl MT Buffer.
2. Secure tubes in FastPrep® Instrument and process at speed 5.5, culture for 30 seconds
and soil for 2 times 30 seconds, with cooling down inbetween. See instrument instructions.
3. Centrifuge Lysing Matrix E Tubes at 14,000 xg for 4 minutes.
4. Transfer supernatant to a clean 1.5 ml eppendorf tube. Add 250µl PPS reagent and mix by
shaking the tube by hand 10 times.
5. Centrifuge at 14,000 xg for 5 minutes to precipitate the pellet. Transfer supernatant to a
clean 4.0ml or 15ml tube. (Resuspend Binding Matrix Suspension before use.) Add 1ml
Binding Matrix Suspension to the supernatant.
6. Place on a rotator (1000rpm) or invert by hand for at least 10 minutes to allow binding of
DNA to matrix.
7. Let stand for 5 minutes, remove 500 µl.
8. Resuspend Binding Matrix in the supernatant. Transfer approximately 600µl of the
mixture to a SPIN Filter and centrifuge at 14,000 xg for 1 minute. Empty the catch tube
and add the remaining supernatant to SPIN Filter and spin again.
9. Add 500µl SEWS-M to the SPIN Filter mix well and centrifuge at 14,000 xg for 1 minute.
Decant flow-through and replace SPIN Filter in Catch tube. Centrifuge at 14,000 xg for 2
minutes to “dry” the matrix of residual SEWS-M wash solution
10. Remove SPIN Filterand place in fresh kit-supplied Catch Tube. Air dry the SPIN Filter
for 5 minutes at room temperature.
11. Add 100µl DES (DNase/Pyrogen Free Water) and gently stir matrix on filter membrane
with a pipette tip or vortex/finger flip to resuspend the silica for efficient elution of the
DNA. Centrifuge at 14,000 xg for 1 minute to transfer eluted DNA to Catch Tube. DNA is
now application-ready.
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From Sample To DGGE Page 3 of 8
PCR with bacteria-specific primers for DGGE
This procedure describes PCR with bacteria-specific primers as these are most generally used.
Of course procedure can be adjusted to use any primer set you like.
Procedure:
1. Calculate first how many samples you wish to amplify, include a negative control (just
water) and one positive control (a marker for DGGE; it is best to include for every 6
samples 1 positive control , the M12 Marker for DGGE). Add one to this number, as a
kind of correction for pipetting errors.
2. For these X pcr-tubes in total prepare a pre-mix using the ‘Muyzer’ primers (AEM 59,
695 (1993)).
Gc clamp
CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC C
F357GC
CGC CCG CCG CGC GGC GGG CGG GGC GGG GGC ACG GGG CCT
ACG GGA GGC AGC AG
ATT ACC GCG GCT GCT GG
R518
3.
Mix (amount for one PCR reaction, multiply by X, prepare the pre-mix and then divide 24 ul over X-1
PCR tubes)
If using standard Taq:
primer F357 (0.01mM stock)
primer R518 (0.01mM stock)
dNTPs (10mM stock)
BSA (biolabs, 10mg/ml)
Taq buffer (10* stock; 2)
Taq enzyme (1)
MQ
total volume
If using Master Mix (FideliTaq):
primer F357 (0.01mM stock) 1.0 µl
primer R518 (0.01mM stock) 1.0 µl
BSA (biolabs, 10mg/ml)
1.0 µl
Master Mix (Fidelitaq)
12,5 µl
MQ
8,5 µl
1.0 µl
1.0 µl
1.0 µl
1.0 µl
2.5 µl
0.50 µl
17 µl
24 µl
total volume
24 µl
4. After dividing the pre-mix over the X-1 tubes
5. Add DNA template (undiluted), 1.0 µl
, to the PCR vial, pipette through the oil
layer.
6. Place the samples in the thermocycler and start the PCR, using the following program:
Time Delay File
Initial denaturation 94 °C 4 min
Step Cycle File
Denaturation
Annealing
Elongation
94°C 0.5 min
54 °C 0.5 min
72°C 0.5 min
35 cycles
Time Delay File
Final elongation
72 °C 5 min
Soak File
cooling
4 °C
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October 17, 2013
15 min
From Sample To DGGE Page 4 of 8
7. Check the results of the PCR by agarose electrophoresis. Mix 5 µl PCR-product, with 1
µl loading buffer and run on an 1.5% agarose gel. Use a 100 bp ladder. You should
expect a product of about 0.2 kb large.
Agarose gel electroforese
Reagents:
- 1* TAE running buffer prepared from 50x concentrated stock solution
- Loading buffer (6X Orange G DNA Loading Dye)
1 Assemble the gelholder. Dissolve 1.0 g agarose in 100 ml TAE (=1.0% agarose gel) in a
microwave or by boiling in an autoclave. When the agarose has dissolved, let it cool to
about 60-80oC, after which you add 1 µl ethidium bromide solution (mutagenic; use
gloves from now on) and pure the gel. Let the gel solidify during 30 minutes. Fill the
electrophoresis tank with TAE buffer and remove the comb.
2 Mix 5 µl DNA template with 1 µl loading buffer.. Place the mixture in one of the wells
and put a DNA molecular weight marker in a well most on the outside.
3 Connect the electrophoresis tank to the power supply. Electrophorese for 15 - 30 minutes
at 100-150 V.
4 Observe the results under UV light (302 nm) with a UV transilluminator, and
document/record the results. Take care: avoid looking directly into UV light, use
protective, transparent shielding, or wear glasses.
DGGE (denaturing gradient gel electrophoresis)
chemicals:
-
Formamide 100%
40% Acrylamide/Bis 37.5:1 (refrigerator)
May Cause Cancer, Toxic !!!!
Ureum
TEMED (N,N,N',N'-tetramethylethylenediamine) (refrigerator)
APS (Ammonium Persulphate)
Tris base (tris(hydroxymethyl)aminoethane
Acetic acid anhydrous 100%
EDTA (ethylenediamine tetraacetic acid)
NaOH
bromophenol blue
sucrose
Ethidium bromide
SDS (sodium dodecyl sulphate)
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Solutions:
-10% APS
make a 10% (w/v) solution in milliQ (1 gram in 10 ml) and
store 0.5 ml alliquots at -20°C.
-50* TAE buffer
242g Tris base; 57.1 ml glacial acetic acid; 100 ml 0.5M EDTA
(pH 8) per liter
-1* TAE running buffer
Mix 20 ml 50* TAE with 980 ml H2O
-Gel-dye
0.05 g bromophenol blue in 10 ml 1* TAE
-Loading buffer (6*)
0.05% (w/v) bromophenol blue (0.05 g)
40% (w/v) sucrose (40 g)
0.1 M EDTA pH=8 (20 ml 0.5M EDTA)
0.5% (w/v) SDS (0.5 g)
adjust volume to 100 ml
-Ethidium bromide
10 mg ethidium bromide in 1 ml H20
-DGGE
0% denaturant solution
Gel percentage
8%
40% acrylamide/Bis
50* TAE buffer
MilliQ
20 ml
2 ml
78 ml
-DGGE
30% denaturant solution
Gel percentage
8%
40% acrylamide/Bis
50* TAE buffer
Formamide
Ureum
MilliQ
20 ml
2 ml
12 ml
12.6 g
to 100 ml
-DGGE
55% denaturant solution
Gel percentage
8%
40% acrylamide/Bis
50* TAE buffer
Formamide
Ureum
MilliQ
20 ml
2 ml
22 ml
23.1 g
to 100 ml
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From Sample To DGGE Page 6 of 8
Optional, but not recommended (in case you want to be flexible in creating another percentage
gel; by mixing 0 and 100% solutions, note however that 100% solutions are prone to
precipitation and introduce additional steps in DGGE preparation which may interfere with
reproducibility):
-DGGE
100% denaturant solution
Gel percentage
8%
40% acrylamide/Bis
50* TAE buffer
Formamide
Ureum
MilliQ
20 ml
2 ml
40 ml
42 g
to 100 ml
Pouring the gel (step 1 and 2 need some time to warm up, ca. 0.5 h)
1. Set up the gradient mixer, on a magnetic stirrer, position well (each time in the same
way) and connect to pump.
2. Turn on the stirrer of the gradient mixer. Check whether tubing and gradient mixer are
not blocked by polymerized acrylamide by pumping around water. Set pump rate to 5
ml/minute (set ‘20’ on Watson Marlow pump).
3. Use gloves from this stage. Clean the glass plates with water and soap, rinse with alcohol.
4. Place the large plate down first, place the 1 mm spacers and on top the short plate.
5. Loosen the screw of the sandwich clamp and place each clamp to the appropriate side of
the gel (arrows facing up and toward the glass plates.
6. Place the sandwich assembly in the alignment slot (slot without cams) of the casting
stand.
7. Insert the alignment card to keep the spacers parallel and push the plates and spacers
down and to each other at the same time you tighten both clamps (not to tight).
8. Check that the plates and spacers are flush at the bottom and tighten the clamp until it is
finger-tight.
9. Place the sandwich on the gray sponge into the casting stand and turn the handles down
to lock the assembly in place. Find the comb you want to use and place it near the stand.
10. Check whether pump and stirrer are working properly (in case you did not earlier, which
actually you should have done)
11. Thaw the APS (500 µl)
12. When you are not using original Biorad glass plates, leakage will often occur. To avoid
this, mix 1 ml of the 55% solution with 5 ul APS and 1 ul TEMED, and add this directly
to the assembled gels. Let it solidify for 30 minutes.
13. Pipette 10.5 ml of the lower and upper gradient solutions in separate tubes (preferably
glass, some types of synthetic material have negative effects on casting of gels). Also
prepare a tube with 5 ml of 0% solution for the stacking gel.
14. The steps below are time-sensitive (7-10 min) so have everything in place. Empty the
gradient mixture and stop the pump. Make sure again that the stirrer is stirring well.
Close the valve between the two chambers of the gradient mixer. Put the needle,
connected via tube to the gradient mixer in the middle of the assembled gel, make sure it
does not fall off easily.
15. Add 50 µl APS to the low and to the high density mix.
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From Sample To DGGE Page 7 of 8
16. Add 10 µl TEMED to the high percentage mixture, invert the tube several times and pour
all solution into the chamber marked with H. This is the chamber that is connected to the
tubing going to the pump. After addition of TEMED polymerization starts so work
relatively fast.
17. Add 10 µl TEMED to the Low mixture, invert and empty into the chamber marked with
L.
18. Open the valve between the two chambers and then turn on the pump. Witness that liquid
is delivered to the assembled gel and that no rapid leakage occurs, during the first minute.
Pumping takes about 5 minutes. In the meanwhile prepare the 0% solution. Add 35 µl
APS. Also add 100 ul of blue loading buffer (to visualize the wells better during loading).
19. After pumping has finished, close the valve between the 2 chambers of the gradient mixer
and fill the L chamber with demi. Put the needle on the outside of the assembled gel and
insert the comb. Add 5 µl TEMED to the 0% solution and add directly to the H chamber.
Now the stacking gel is poured. Wait until solution pours over from the assembled gel.
Disconnect the needle and open the valve of the gradient mixture in order to clean the
mixer with water by pumping water through the system
20. Let the gel polymerize 1.5 - 2 hours. When running the next day, wrap in plastic (after
waiting for 2 hrs) and store at 4oC.
Running DGGE gels (usually the next day)
1. Fill the electrophoresis tank with 7 liter of fresh 1* TAE running buffer, prepared freshly
from 140 ml 50 x TAE buffer.
2. Place the temperature control module on top of the tank, attach the power and turn the
power, the pump and the heater on
3. Set the temperature controller to the desired temperature (60°C for DGGE) and the ramp
rate to 200°C/hr
4. Preheat the buffer to the set temperature, it can take 1 to 2 hours for the system to heat
the buffer up to the desired temperature
5. Turn the control unit off and remove it, just prior to running the gel and when the tank is
at the right temperature
6. Take out 500 ml running buffer.
7. After polymerization, remove the comb from the gel by pulling it straight up slowly and
gently.
8. Release the gel from the casting stand.
9. Hold the gel on its side, above the sink, clear the wells from non-polymerized acrylamide
by rinsing it with 60oC hot running buffer (use a 60 ml syringe, be careful not to damage
the wells) so that the buffer can flow out of the well.
10. With the short plate facing the core, slide the sandwich in place, push the gel onto the
core until you hear a click.
11. Place a sandwich to the other side or use a set off glass plates without spacers to
complete the upper chamber (do not forget).
12. Pour 300 ml buffer into the upper chamber to check if it is leaking.
13. Place the attached gel assemblies into the buffer chamber by positioning the red button at
the right hand side and replace the control module on top of the tank.
14. Turn the unit on again.
15. Allow the system to reach the initial temperature (60oC) and check if the system is well
connected by supplying power to the gel (let it run for 1 min, to check for possible errors,
which at this stage you can still easily fix).
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From Sample To DGGE Page 8 of 8
16. Prepare the samples (4 µl loading buffer + 20 µl sample (for clones 1-2 ul is sufficient,
for marker 10 µ))
17. Load the gel by using the special gel saver tips. Mark the gel by not using one or more
wells. For high quality gels, do not use the outer 2 wells. So use maximally 16 samples.
For Gelcompar gel analysis; use marker in lane 3, 8, 13 and 18.
18. Run for 4 hours 200 Volt, and 60°C
19. After running turn off the power supply, remove the control unit and take out the core.
20. Remove the sandwich and the clamps and remove one glass plate.
21. Cut off an edge of the gel in order to remember its position.
22. Put the gel on the glass plate in running buffer containing 1 µl per 100 ml Ethidium
bromide, or 20 µl SYBR Green I (Sigma) in 200 ml TAE.
23. Stain for 20 (SYBR Green) - 60 minutes (EtBr) and place the gel on a UV transparent
plate on a UV transilluminator,
24. Make a picture and store your image on a diskette or scan the photo for gel analysis.
SampleToDGGE.docx
October 17, 2013