From Sample To DGGE Page 1 of 9

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1 From Sample To DGGE Page 1 of 9 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 ).

2 From Sample To DGGE Page 2 of 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.

3 From Sample To DGGE Page 3 of 9 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 the M12 Marker; it is best to include for every 6 samples 1 positive control). 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)). F357 F357-GC R518 CCT ACG GGA GGC AGC AG CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CAG ATT ACC GCG GCT GCT GG 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: If using Master Mix (FideliTaq): primer F357 (0.01mM stock) 1.0 µl primer F357 (0.01mM stock) 1.0 µl primer R518 (0.01mM stock) 1.0 µl primer R518 (0.01mM stock) 1.0 µl dntps (10mM stock) 1.0 µl BSA (biolabs, 10mg/ml) 1.0 µl BSA (biolabs, 10mg/ml) 1.0 µl Master Mix (Fidelitaq) 12,5 µl Taq buffer (10* stock; 2) 2.5 µl MQ 8,5 µl Taq enzyme (1) 0.50 µl MQ 17 µl total volume 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. 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 94 C 0.5 min Annealing 54 C 0.5 min Elongation 72 C 0.5 min 35 cycles Time Delay File Final elongation 72 C 5 min Soak File cooling 4 C 15 min

4 From Sample To DGGE Page 4 of 9 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 o C, 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 minutes at 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) Introduction DGGE is an electrophoretic separation method based on differences in melting behaviour of double stranded DNA fragments (Fisher and Lermann, 1979). The electrophoresis takes place in a vertically placed polyacrylamide gel in a gradient of denaturants. The quality of the DGGE is determined by the quality of the PCR products. Double bands most often are a PCR problem, not an electrophoresis problem. We use the DCODE system of BIORAD It is important that the buffer in the upper buffer compartment is circulated to prevent exhaustion of buffer components. In addition the gel should be kept at constant homogeneous temperature (we use 60 C). To accomplish this we place the gel(s) in a compartment containing the buffer. Buffer is circulated by a pump to the upper buffer compartment and via an overflow it is circulated back. In the DCODE system, the mixing of the buffer (and temperature homogeneity) is improved by using a magnetic stirring bean, driven by a stirrer which is placed under the DCODE system. To set up a new DGGE (new primer set, new sample type/habitat) we usually start with a relatively broad gradient (20-80% denaturant). However, we soon focus to the area of interest which should include the highest and lowest bands in different samples. For the Eukaryotic DGGE described by Van Hannen et al. (1998) as well as for the bacterial DGGE described by Muyzer et al. (1993) we use a denaturant gradient of 30 to 55%. The gel is 1.5 mm thick and contains 8% polyacrylamide.

5 From Sample To DGGE Page 5 of 9 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) 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 H 2 O -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 H 2 0

6 From Sample To DGGE Page 6 of 9 -DGGE 0% denaturant solution Gel percentage 8% 40% acrylamide/bis 20 ml 50* TAE buffer 2 ml MilliQ 78 ml -DGGE 30% denaturant solution Gel percentage 8% 40% acrylamide/bis 20 ml 50* TAE buffer 2 ml Formamide 12 ml Ureum 12.6 g MilliQ to 100 ml -DGGE 55% denaturant solution Gel percentage 8% 40% acrylamide/bis 20 ml 50* TAE buffer 2 ml Formamide 22 ml Ureum 23.1 g MilliQ to 100 ml 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 20 ml 50* TAE buffer 2 ml Formamide 40 ml Ureum 42 g MilliQ to 100 ml

7 From Sample To DGGE Page 7 of 9 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 Demi. 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 100% 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. 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.

8 From Sample To DGGE Page 8 of Turn on the pump and open the valve between the two chambers. Witness that liquid is delivered to the assembled gel and that no rapid leakage occurs, during the first minute. Pumping takes about 5 minutes. 19. 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). 20. 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 21. Let the gel polymerize hours. When running the next day, wrap in plastic (after waiting for 2 hrs) and store at 4 o C. Running DGGE gels (usually the next day) Prepare electrophoresis tank one day in advance and use the timer to start it on time for preheating the buffer (step 1 4). 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 60 o C 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.

9 From Sample To DGGE Page 9 of 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 (60 o C) 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). 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 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.