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DNA stable isotope probing (DNA-SIP)
DNA stable isotopes is a cultivation-independent method for identifying and characterizing active communities of microorganisms that are capable of becoming certain substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labeled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labeled DNA for downstream molecular analysis.
DNA stable isotopes (DNA-SIP) is a powerful method for identifying active microorganisms, particularly carbon substrates and assimilating nutrients into cellular biomass. As such, this cultivation-independent technique has an important methodology for assigning metabolic function to the various communities inhabiting a variety of terrestrial and aquatic environments. After incubation of an environmental sample with stable isotopically labeled compounds, nucleic acid extracted density gradient ultracentrifugation and then subjected to gradient fractionation in order to separate nucleic acids of different densities. Purification of DNA from cesium chloride retrieves labeled and unlabeled DNA for subsequent molecular characterization (e.g. fingerprints, microarrays, clone libraries, metagenomics). This JoVE Video Protocol provides step-by-step visual explanations for the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labeled DNA. The log also contains sample SIP data code and highlights important tips and hints that must be considered in order to ensure a successful DNA SIP analysis.
1. Prepare the reagents
DNA-SIP requires the use of reagents, which should be prepared in advance of the actual procedure. The instructions for preparing each reagent are listed in this section and are taken from an earlier SIP protocol 1 changed.
- Cesium Chloride (CsCl) Solution for Preparing SIP Gradients - Prepare a 7.163 M CsCl solution by gradually dissolving 603.0 g of CsCl in distilled and deionized water (ddH 2 O) to a final volume of 500 mL. Be careful not to exceed 500 mL! Warming the solution slightly while stirring will help dissolve all of the CsCl. Aliquot the final solution in sealed aliquots. In our laboratory, a common storage practice is to prepare 100-ml aliquots in 125-ml serum vials, which are then crimp-sealed with butyl rubber stoppers. The sealed aliquots can be stored indefinitely at room temperature (20 ° C). The seals prevent evaporation and CsCl "crust" formation. Determine the density of the solution by weighing three-fold 100-μl aliquots, or using a digital refractometer (e.g. Reichert AR200) that has been carefully calibrated for CsCl solutions. After the calibration is successful, the Reichert AR200 becomes consistent and delivers precise measured values for several years. At room temperature (20 ° C), the final density of this solution is typically in the range of 1.88 to 1.89 g ml -1. The density varies slightly every time a new camp is prepared.
- Cesium chloride solution preparing gradient with ethidium bromide (EtBr) - Combine 250 g of CsCl with 250 ml of sterile ddH 2 O water. Aliquot this solution into separate serum vials that have been crimp-sealed with butyl rubber seals as described in 1.1.
- Gradient Buffer - Combine 50 ml of 1 M Tris-HCl, 3.75 g KCl and 1 ml 0.5 M EDTA in 400 ml water. Dissolve the KCl, then add the ddH 2 O to 500 ml. Filter-sterilized and autoclave. The final solution is 0.1 M Tris, 0.1 M KCl, and 1 mM EDTA.
- Polyethylene Glycol (PEG) Solution - Prepare the PEG solution by dissolving 150 g of polyethylene glycol 6000 and 46.8 g of NaCl in sterile ddH 2 O water to a total volume of 500 mL (30% PEG, 1.6 M NaCl). Autoclave.
Note: This solution separates into two phases with autoclaving. Add a stir bar to the autoclaved bottle so that the solution can be properly mixed when it does.
- TE buffer - Prepare a solution of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0) in sterile ddH 2 O water, with autoclaved stock solutions of 1 M Tris-HCl (pH 8.0) and 0.5 M EDTA (pH 8.0). Sterilize filter and autoclave.
- 70% Ethanol - Combine 350 ml of high purity ethanol with 150 ml of sterile ddH 2 O water.
2. Sample incubation and DNA extraction
For the DNA-SIP incubations, the samples are usually made with heavy isotope carbon (13 C) incubated. Incubation times and conditions (e.g. nutrient supplementation, moisture, light) depend on the type of sample being incubated and the nature of the substrate. DNA-SIP experiments have been carried out successfully with a wide variety of single carbon compounds 2,3, multi-carbon compounds 4,5,6 and labeled with nitrogen or oxygen 7,8 9. However, there is a downside to using 15 N-or 18 O-labeled compounds of the decreased physical separation of the labeled nucleic acid, primarily due to the presence of fewer nitrogen and oxygen atoms in DNA and RNA relative to carbon atoms.
A critical control point for the DNA-SIP experiments is an identical incubation with native (e.g. 12 C) substrate. This incubation provides a subsequent comparison to ensure that obvious labeling of nucleic acid is not an artifact of ultracentrifugation or that G + C content density differences in DNA contribute to the separation 10. It is also important to keep frozen sample material for comparison with "light" and "heavy" DNA, and to assess the value with a non-substrate control in the background to assess changes throughout the SIP incubation.
- Incubate environmental samples in microcosms with labeled substrate (Figure 1). In our experience we have found that a minimum inclusion is between 5-500 pmol 13 C carbon per gram of sample will be used for samples with high biomass such as soil samples 1. For samples living in water with less biomass than soil, 100 to 100 pmol can be integrated 13 C carbon per liter result in a detectable heavy isotopic signature 1. The amount of carbon change will depend on the percentage of carbon in biomass and the requirement for the additional nutrient supplement for assimilation integrated all on the properties of the samples analyzed and the targeted organisms of interest. A single set of sample incubation guidelines do not apply to all samples. It is important that the substrate concentration used for SIP incubation ideally be as close as possible to the concentration normally used in situ encountered; Experimental bias can be a result of enrichment culture conditions 10 be.
- After incubating the sample with stable isotopically labeled substrate, extract DNA from microcosms using a rigorous extraction protocol (PCR or small insert cloning) or a trusted enzymatic lysis for high molecular weight cloning (e.g., large insert metagenomics). RNA co-extraction usually does not affect analysis, so protocols that yield RNA as well as DNA can be used. The ultracentrifugation of the extracted DNA will not shear fragments shorter than ~ 50 kb pairs 1.
- Quantify extracted DNA prior to the CsCl gradient ultracentrifugation tube setup. Quantify the DNA with a spectrophotometer (e.g. Nanodrop 2000) if the extraction protocol only delivers DNA (e.g. column-based kits). Alternatively to be quantified using agarose gel electrophoresis.
3. Prepare gradient solutions for ultracentrifugation
This procedure involves adding DNA to ultracentrifuge tubes. There is more than one type of hose and rotor so the exact protocol will vary and depend on the manufacturer's instructions. That is, we recommend the use of a vertical rotor also to ensure the maximum possible separation of light and heavy DNA. We use a Beckman-Coulter Vti 65.2 rotor with 16 wells to accommodate 5.1 ml QuickSeal Polyallomer tubes and the protocol will ensure the steps and considerations for these conditions.
- Using the DNA concentrations determined in step 2.3, calculate the required amount of extracted DNA that will be required to add 0.5 ug - 5 ug of DNA in the ultracentrifuge tubes.
- Combine extracted DNA (0.5 to 5 ug) with Gradient Buffer (see step 1.3) and 4.8 ml of 7.163 M CsCl to a total volume of approximately 6 ml in a sterile, disposable 15 ml tube. Note that the density of the CsCl solution can also vary with the same molarity (see step 1.1). The following equation can be used to determine the volume of Gradient Buffer / DNA mixture required to create an appropriate mix ratio:
Gradient buffer and DNA solution (ml) = (CsCl stock solution density - desired final density) x volume of CsCl stock solution x 1.52
Enter the volume of the CsCl stock solution at 4.80 ml. The desired final density should be 1.725 g ml -1. The stock solution density was determined in step 1.1.
Also note that the relative volume of CsCl and Gradient Buffer / DNA will result in a total volume greater than 5.1 ml. Preparing volumes greater than the maximum volume capacity of the ultracentrifuge tubes (greater than 5.1 ml) will ensure that there is enough solution to completely fill the tube.
- Mix by inverting 10 times. DNA becomes stable in CsCl at room temperature.
4. Create an EtBr control gradient (optional)
Since EtBr is an intercalating dye complex with DNA that makes it visible under UV light, control gradients with EtBr are helpful because they provide immediate visual confirmation of gradient formation prior to fractionation of sample tubes (e.g. see Figure 1). Including a head tube with EtBr and a mixture of both 12 C-DNA and 13 C-DNA (or 14 N-DNA and 15 N-DNA) enables the band formation within the tubes to be visualized immediately after the ultracentrifugation is complete. This is important because a broken tube during ultracentrifugation or incorrectly programmed run conditions can result in failed gradient formation. When bound to DNA, EtBr lowers the density of the DNA and as a result another protocol is to be prepared followed by gradients. Note that other nucleic acid stains instead of EtBr 11 can be used but that may require protocol optimization with other fluorophores.
- The control gradient requires two volumes of genomic DNA: one fully labeled with stable isotopes and one without a label. We usually use either Sinorhizobium meliloti in media with 13 C or 12 C-glucose as the only carbon source or Methylococcus capsulatus Tribe of Bath in the presence of 13 C or 12 C-methane bred, cultured as our controls.
- Combine a 5 -10 pg amount of both of the 12 C-DNA and 13 C-DNA with gradient buffer to a final volume of 1.00 ml in a disposable 15-ml screw-cap tube.
- Add 1.00 g of solid CsCl to the same tube. Mix by inverting.
- Add 110 ul to 10 mg ml -1 EtBr solution and 4.3 ml of a 1 g ml -1 CsCl stock solution to the same screw cap tube used in step 4.2. The final density of the solution will be roughly that of the original CsCl stock solution.
- An additional "blank" control solution with EtBr will also be required to counterbalance the solution created in step 4.4. Combine 1.00 mL of the Gradient Buffer, 1.00 g of CsCl, 110 μl of a 10 mg ml -1 EtBr solution and 4.3 ml of a 1 g ml -1 Put CsCl stock solution in a separate 15 ml screw cap tube and mix by inversion.
- Using a light bulb and Pasteur pipette, carefully fill in the ultracentrifuge tubes with gradient solutions prepared in step 3.2 (or step 4.4 if preparing an EtBr control gradient). Carefully apply the solutions to the tubes with a Pasteur pipette. Label the tubes on the tube shoulder with a fine felt-tip pen. CAUTION: Make sure the tubes are filled exactly to the bottom of the tube neck. Insufficiently filled tubes are likely to burst during ultracentrifugation.
- When all the required tubes are filled with sample solutions, write down the exact dimensions of each tube. Couple tubes and balance them on, within 0-10 mg. For balancing, find almost pairs and add or remove tiny amounts of the solution until they are balanced, keeping the solution on as close to the bottom of the tube's necks as possible. Note that for weighing vessels we use an inverted 15-ml screw-top tube that was cut in half as a tube holder for balance.
- Seal the tubes with a "tube topper" according to the manufacturer's instructions.
- Check that the pipes are properly sealed by inverting them and applying moderate pressure. Weigh the tubes again to check that they are still weighed within 0-10 mg after sealing.
- Also inspect each rotor carefully to make sure the well is clean and free of any debris or dust that might puncture the tubes during ultracentrifugation.
- Place the tubes in the rotor with the balanced pairs facing each other. Record the rotor location of each sample because the ultracentrifugation process can cause marker labels to be damaged or erased. Carefully close the rotor wells as directed by the manufacturer.
- Place the rotor in the ultracentrifuge. Close the ultracentrifuge door and apply a vacuum. If using a Vti 65.2 rotor, set the speed to 44,100 revolutions per minute (~ 177,000 xg av), the temperature at 20 ° C, and ultracentrifugation time for 36-40 hours. Select vacuum, maximum acceleration, and turn on the brakes (ensures gradients are not interrupted by deceleration). Note that turning off the brake will add an additional 1-2 hours to the run time. Also note that shorter durations may not provide adequate tape resolution. Long ultracentrifugation runs are recommended as they result in a higher resolution of different nucleic acid bands.
- Immediately after completing the ultracentrifugation procedure, carefully remove the rotor. To avoid any tilting or bumping of the rotor, remove tubes from the rotor to prevent the incline inside the tubes. In rare cases, a tube will burst during the run. If so, there is a chance that the gradients in the other tubes are not forming properly. When a gradient control has been added, inspect this tube carefully under UV light to confirm gradient formation. If the slope is not properly formed in the head tube, it is best to repeat all of Step 5. Note that the EtBr head tube and its blind control can be stored and reused in the dark for up to six months. Make sure the rotor is clean according to the manufacturer's instructions after the burst tube has been removed. Do not use metal brushes or abrasives to clean the rotor fountain to avoid scratching the rotor fountain! Rotor-specific brushes and cleaning supplies can be purchased from Beckman.
6. Gradient fractionation
There are two methods currently used to recover DNA from ultracentrifuge tubes: fractionation and needle extraction. This protocol will only describe the process of obtaining DNA using the fractionation technique. This is because, for most SIP experiments, labeled DNA cannot be visualized with EtBr and must instead be identified by comparing the corresponding light and heavy fractions from several sample tubes. A syringe pump is strongly recommended to retrieve density gradient fractions from ultracentrifuge tubes. We work with a BSP model infusion pump (Braintree Scientific Inc.). A low-flow peristaltic pump or an HPLC pump can also be used.
- Fill a sterile 60 ml syringe with sterile ddH 2 O contains sufficient bromophenol blue dye to give it a dark blue color. Place the syringe on the loading arm of the syringe pump. Attach pump tubing with a 23-lane 1 "needle and turn on the pump until some ddH 2 O to the end of the needle. Note that any air bubbles that may be present in this ddH 2 O supply will have a negative impact on the disassembly has come built-in.
- Fix one of the ultracentrifugation tubes to a clamp stand. Make sure that the clamp is tight enough to prevent the tube from being dislodged, but not so that pressure on the tube would cause a release of the CsCl solution when the tube is punctured. Pierce the very bottom of the tube along the tube suture with a fresh 23 gauge 1 "needle. For best results, pierce the tube, in a controlled, quick, and safe manner.This very difficult to do well is to practice several times before attempting this with sample tubes first.
- Prepare 12 sterile 1.5 ml reaction vessels for each sample, identify the number and proportion of the sample (1-12; difficult to easy). Using the needle around the pump tubing (step 6.1), pierce the top of the tube on the top tube shoulder, along the suture. Collect the gradient solution using the microcentrifuge tube. As for the lower part of the tube, piercing the tube is done in a quick and controlled manner. Practice beforehand and be very careful to use a controlled pulling motion to prevent the forced needle from going through the tube and into a finger! Using a previously calibrated pumping rate that produces 12 x 425 μl fractions in 12 minutes (425 μl min -1) he brings.
- Use a digital refractometer (e.g. Reichert AR200; recommended) or an analytical balance to check the density of the fractions from a slope to confirm proper gradient formation. You need to use ~ 50 µl sample for the test. We have often heard pure culture DNA in a tube (as described for the preparation of the EtBr control gradient) to serve as a control for fractionation and use this for density determination. Expect to densify ~ 1.690-1.760g ml -1 Area, with an average density of ~ 1.725 g ml -1.
7. DNA precipitate
- Precipitate DNA from all fractions by first adding 20 µg of linear polyacrylamide as a support for the precipitation. Mix by inverting. Add 2 volumes of PEG solution (see step 1) and mix by inversion. Note that a support for precipitation (e.g. glycogen or linear polyacrylamide) is critical for quantitative recovery of DNA from gradient fractions, but caution should be exercised when using glycogen as a support for precipitation for this protocol. Glycogen preparations have shown that bacterial nucleic acids can be contaminated and impurities can easily confuse the interpretation of the SIP gradient fractions 12.
- Leave the tubes at room temperature for 2 hours in order to be able to precipitate the DNA. If desired, tubes can be left at room temperature overnight.
- Centrifuge at 13,000 g for 30 minutes with the back of the tubes facing out for consistent tube orientation in the rotor. Aspirate carefully and discard the supernatant. A pellet should be visible but can be very difficult to see at this stage. Working under a bright light source (e.g. desk lamp) to assist in the visualization of the pellets.
- Wash the pellet with 500 µl 70% ethanol. Centrifuge at 13,000 g for 10 minutes. Carefully aspirate and discard the supernatant. The pellet will usually be more visible for this step, but will more easily distance itself from the pipe wall.
- Let the pellet dry at room temperature for 15 minutes.
- Suspend each pellet in 50 μl of TE buffer (see step 1.5). Run 5 μl of each fraction on an agarose gel according to standard laboratory protocols.
8. Fraction characterization
The method used to assess gradient fractions to characterize the success of a SIP incubation will vary depending on the laboratory and equipment availability. Using a fingerprint method for the alignment of the 16S rRNA gene is a common approach and methods such as terminal restriction fragment length polymorphism (T-RFLP) or denaturing gradient gel electrophoresis (DGGE) are appropriate (Figure 1). Following the protocol described above, expect the light DNA to be found with fractions 9-11 (~ 1.705-1.720 g ml -1) and the severe DNA fingerprints have been linked to within fractions of 5-8 (~ 1.720-1.735 g ml -1 be associated). Unique fingerprints associated with fractions 5-8 of stable isotopic incubated samples but not incubated with native substrate controls provides strong evidence that certain organisms are involved in the metabolism of particular labeled substrate. If not enough labeled DNA is left for some applications (hybridization, metagenomics), multiple displacement amplification can be used to produce larger quantities from 13 to 15 but this can be chimeric in the amplified DNA 14,16 to introduce.
Typical DNA-SIP results will show a separation of labeled and unlabeled DNA in the gradients formed by ultracentrifugation. Ideally, a complete regression of the high molecular weight genetic material (e.g. 13 C, 15 N) can be achieved by unmarked materials. Dissolution can be observed visually by observing tape formation in EtBr tubes. The concentrations of the retrieved genomic DNA contained in each gradient fraction can also be used to confirm proper gradient formation.
For this protocol we are representative of the results of the ultracentrifugation done with nucleic acid from two pure cultures (Figure 2). The fractionated gradient presented here was made using genomic DNA S. extracted meliloti (ATCC 1021) and 13 C-labeled M. capsulatus st. Bath. After ultracentrifugation, fractionation and DNA recovery, labeled and unlabeled genomic DNA are separated into the respective gradient fractions with different densities (Figure 2A). Heavy isotope of labeled DNA can be seen in fractions 4-5, while unlabeled DNA is found in high concentrations in fractions 9-10. The DNA from each fraction was denatured using gradient gel electrophoresis 17 and the PCR-amplified products generated discrete banding patterns corresponding to the two organisms contained in the slope (Figure 2B) characterized. The density of the fractions ranged from ~ 1.580 to 1.759 g ml -1, and they are shown in order of decreasing density from left to right.
Though the separation from pure 13 C-and 12 C-DNA can be spoken (Figure 2), environmental sample incubations can be more difficult to interpret. For example, we incubated with tundra soils from Resolute Bay (Nunavut, Canada) 12 C or 13 C-labeled glucose for a 14-day period at 15 ° C. The agarose gels of purified gradient fraction DNA demonstrated that genomic DNA was "smeared" over fractions 7-10 for both 12 C and 13 C incubations (Figures 3A and 3C, respectively). In this case you can 13 C-enrichment of biomass from certain microbial taxa only with an approach such as DGGE can be determined from 16S rRNA genes. The 12 C-glucose incubated soil DNA produced similar patterns in all gradient fractions (3B), but the 13 Incubated C-glucose sample generated DGGE fingerprints that are clearly associated with fractions 5-8 (Figure 3D). Of particular interest are the conserved bands indicated by the arrows. The dominant "phylotype" is in all gradient fractions but heavier fractions for the DNA shift from 13 C-glucose incubated soil obtained. Subsequent DNA sequencing of this tape and / or clone library analysis would confirm the identity of this particular 16S rRNA gene and subsequent guidance on metagenomic or cultivation-based approaches.
Illustration 1. A DNA-SIP experiment with sample incubation, DNA extraction, CsCl density gradient ultracentrifugation and DNA characterization with molecular techniques outline.
Figure 2. Expected results for SIP gradient fractionation including DNA from two pure cultures. (A) Aliquots of DNA from gradient fractions 1-12 were made from a gradient on a 1% agarose gel 13 C-labeled M. to run capsulatus Tribe Bath (fractions 4-6) and 12 C-labeled S. meliloti (Fractions 8-10). A 1-kb ladder is included for comparison (B) PCR amplified DNA from the same fractions were run on a 10% DGGE gel. Fingerprint patterns show clear differences between fractions 5 and 9, for example.
Figure 3. Expected results for the SIP gradient fractionations from soil sample incubations. Aliquots of gradient fractions from both 12 C-glucose changed soil (A) and 13 C-glucose modified soil (C) were run on 1% agarose gels and a 1-kb ladder is included for comparison. Corresponding DGGE fingerprints for each of these samples are in (B) and (D). Fraction fingerprinting reveals accumulation of certain bacterial taxa in the 13 C-glucose changed sample into fractions 5-8 (D).
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Proper construction of the stable isotope experiments is crucial for the preservation of labeled DNA against the background of unlabeled community. Considerations related to incubation times, substrate concentrations, incubation conditions (e.g. nutrients, soil moisture content), cross-feeding, and sample replication have been discussed elsewhere 10,18 and discussed, we encourage the reader to consult these publications when designing a SIP incubation. With respect to the current protocol, it is worth commenting on additional considerations regarding the interpretation of the data from SIP gradients. Due to the nature of the ultracentrifugation process, it is additionally important to include controls such as pure cultures and native-substrate incubated samples to ensure that the bands are shown or hidden in particular fractions, not artifacts of the protocol. For example, DNA in an ultracentrifuge gradient may not be visible in an agarose gel (Figure 2A), but can still contaminate the full length of the gradient (Figure 2B). Although M. capsulatus Patterns are shown most clearly in the dense fractions (5-7) of the gel in Figure 2B, the same DGGE pattern was still observed in the lightest fraction (12). With carefully checked controls, the interpretation of the SIP gradient fraction data is possible.
Due to the nature of some well-designed SIP experiments (e.g., nearby in situ Substrate concentrations, short incubation times), isotope incorporation can be very low 10. In addition, most microorganisms in terrestrial or aquatic environments have long generation times compared to growth in the laboratory, and require longer incubation times to achieve detectable levels of isotope accumulation. Other populations may be able to metabolize a variety of substrates, and cannot grow fully on labeled substrate. There are also communities (e.g., groundwater) that may be associated with low biomass levels and generate low yields of the extracted nucleic acid. In all of these cases, the quantitative retrieval of labeled nucleic acids can be challenging.
To circumvent these restrictions there are a large number of natural and synthetic carrier molecules that assist in the precipitation and utilization of DNA from CsCl gradients. Carrier molecules can be of origin, such as glycogen or DNA, from an archaeal organism 19 or synthetic in nature, such as linear polyacrylamide biological. The advantage of using carrier molecules like these when performing DNA-SIP is that they allow visualization of the bands in the CsCl gradients that would normally not be visible and ensure quantitative recovery of low DNA concentrations. Successful recovery of low nanogram amounts of DNA from CsCl gradients actually requires the use of a carrier molecule 1,12. Recent research has shown that carrier molecules obtained from biological sources are often combined with DNA from the source organism 12 become contaminated and the results are very difficult of patterns with 13 C-labeled DNA (data not shown) linked to differentiate. It is therefore recommended that synthetic carrier molecules such as linear polyacrylamide are used for DNA-SIP. In addition, the use of multiple displacement amplification (MDA) can generate high fidelity yields from labeled DNA for downstream molecular analysis 13,14, although chimeras can be generated by amplification, and recognized in downstream molecular analysis 14.
One of the most powerful uses of DNA-SIP, which has not yet been exhausted, is the possible recovery of DNA from the active community members for metagenomic library analysis. We expect that great advances in enzyme discovery will result from the integration of stable isotopes into existing metagenomic surveys from diverse terrestrial and aquatic environments. The protocol visualized here to produce labeled DNA of sufficient quality for these discovery-based applications.
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No conflicts of interest declared.
This work was supported by Strategic Project and Discovery grants to JDN from the Natural Sciences and Engineering Research Council of Canada (NSERC).
|Bromophenol Blue||Reagent||Fisher Scientific||BP115-25|
|Cesium chloride||Reagent||Fisher Scientific||BP210-500|
|Ethanol, reagent grade||Reagent||Sigma-Aldrich||652261|
|Hydrochloric acid||Reagent||Fisher Scientific||351285212|
|Polyethylene Glycol 6000||Reagent||VWR international||CAPX1286L-4|
|Potassium Chloride||Reagent||Fisher Scientific||AC42409-0010|
|Sodium Chloride||Reagent||Fisher Scientific||S2711|
|Sodium Hydroxide pellets||Reagent||Fisher Scientific||S3181|
|Tris base||Reagent||Fisher Scientific||BP1521|
|Dark Reader||Equipment||Clare Chemical||DR46B|
|Nanodrop 2000||Equipment||Fisher Scientific||361013650|
|Infusion pump||Equipment||Braintree Scientific, Inc.||N / A||Model Number: BSP|
See www.braintreesci.com for ordering details.
|Tube sealer||Equipment||Beckman Coulter Inc.||358312|
|Ultracentrifuge||Equipment||Beckman Coulter Inc.|
|Ultracentrifuge rotor||Equipment||Beckman Coulter Inc.||362754|
|Ultraviolet light source||Equipment||UVP Inc.||95-0017-09||Any UV source will suffice|
|Ultraviolet light face shield||Equipment||Fisher Scientific||114051C|
|Butyl rubber stoppers, gray||material||Sigma-Aldrich||27232|
|Centrifuge tubes||material||Beckman Coulter Inc.||342412|
|Hypodermic needle, 23 gauge, 2 ”length||material||BD Biosciences||305145|
|Microfuge tubes, 1.5 mL||material||DiaMed||AD151-N500|
|Open center seals, 20 mm diameter||material||Sigma-Aldrich||27230-U|
|Pasteur pipettes, glass||material||Fisher Scientific||13-678-6C|
|Pipet tips||material||DiaMed||BPS340-1000||Catalog number is for 200 μl tips. 10 or 20 μl tips may be purchased from the same source|
|Pump tubing 1.5 mm bore x 1.5 mm wall||material||Appleton Woods|
|Screw-cap tubes, 15 mL||material||DiaMed||AD15MLP-S|
|Serum vials, 125 mL volume||material||Sigma-Aldrich||Z114014|
|Syringe, 60 mL||material||BD Biosciences||309653|
DOWNLOAD MATERIALS LIST
- Neufeld, J.D. DNA stable-isotope probing.Nat. Protocols. 2, 860-866 (2007).
- Neufeld, J. D., Boden, R., Moussard, H., Schäfer, H., Murrell, J. C. Substrate-specific clades of active marine methylotrophs associated with a phytoplankton bloom in a temperate coastal environment.Appl. Environ. Microbiol. 74, 7321-7328 (2009).
- Nercessian, O., Noyes, E., Kalyuzhnaya, M. G., Lidstrom, M. E., Chistoserdova, L. Bacterial populations active in metabolism of C1 compounds in the sediment of Lake Washington, a freshwater lake.Appl. Environ. Microbiol. 71, 6885-6899 (2005).
- Padmanabhan, P. Respiration of 13C-labeled substrates added to soil in the field and subsequent 16S rRNA gene analysis of 13C-labeled soil DNA.Appl. Environ. Microbiol. 69, 1614-1622 (2003).
- Bernard, L. Dynamics and identification of soil microbial populations actively assimilating carbon from 13C-labeled wheat residue as estimated by DNA- and RNA-SIP techniques.Environ. Microbiol. 9, 752-764 (2007).
- Haichar, elZ. ahar, F, Identification of cellulolytic bacteria in soil by stable isotope probing.Environ. Microbiol. 9, 625-634 (2007).
- Addison, S., McDonald, I., Lloyd-Jones, G. Stable isotope probing: Technical considerations when resolving 15N-labeled RNA in gradients.J. Microbiol. Meth. 80, 70-75 (2009).
- Buckley, D. H., Huangyutitham, V., Hsu, S. -F., Nelson, T. A. Stable isotope probing with 15N achieved by disentangling the effects of genome G + C content and isotope enrichment on DNA density.Appl. Environ. Microbiol. 73, 3189-3195 (2007).
- Schwartz, E. Characterization of growing microorganisms in soil by stable isotope probing with H218O.Appl. Environ. Microbiol. 73, 2541-2546 (2007).
- Neufeld, J. D., Dumont, M. G., Vohra, J., Murrell, J. C. Methodological considerations for the use of stable isotope probing in microbial ecology.Microb. Ecol. 53, 435-442 (2007).
- Martineau, C., Whyte, L., Greer, C. Development of a SYBR safe technique for the sensitive detection of DNA in cesium chloride density gradients for stable isotope probing assays.J. Microbiol. Meth. 73, 199-202 (2008).
- Bartram, A. K., Poon, C., Neufeld, J. D. Nucleic acid contamination of glycogen used in nucleic acid precipitation and assessment of linear polyacrylamide as an alternative co-precipitant.Biotechniques. 47, 1019-1022 (2009).
- Chen, Y. Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyzes of uncultivated Methylocystis in acidic peatlands.Environ. Microbiol. 10, 2609-2622 (2008).
- Neufeld, J. D., Chen, Y., Dumont, M.G., Murrell, J.C. Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics.Environ. Microbiol. 10, 1526-1535 (2008).
- Kalyuzhnaya, M. High-resolution metagenomics targets specific functional types in complex microbial communities.Nat. Biotechnol. 26, 1029-1034 (2008).
- Binga, E.K., Lasken, R.S., Neufeld, J.D. Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology.ISME J. 2, 233-241 (2008).
- Green, S. J., Leigh, M. B., Neufeld, J. D. Microbiology of Hydrocarbon and Lipid Microbiology. Timmis, K. N. Springer-Verlag. Berlin Heidelberg. 4137-4158 (2010).
- Neufeld, J. D., Wagner, M., Murrell, J. C. Who eats what, where and when? Isotope labeling experiments are coming of age.ISME J. 1, 103-110 (2007).
- Gallagher, E., McGuinness, L., Phelps, C., Young, L. Y., Kerkhof, L. J. DNA shortens the incubation time needed to detect benzoate-utilizing denitrifying bacteria by stable-isotope probing.Appl. Environ. Microbiol. 71, 5192-5196 Forthcoming.
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