Supporting Text. Methods

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Roberta Harris
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1 Supporting Text Methods PCR Amplification and Sequencing of amoa. Each reaction mixture contained 12.5 µl of MasterAmp PCR premix F (Epicentre Technologies, Madison, WI), 0.5 µm of each primer (Qiagen, Valencia, CA), 2.5 units of AmpliTaq DNA polymerase LD (Applied Biosystems), and 0.25 µl of template DNA. Amplification was performed in a total volume of 25 µl in 0.2-ml reaction tubes with a DNA Engine thermal cycler (MJ Research, Cambridge, MA). All PCR was performed in triplicate. PCR products were cloned by using the TOPO TA Cloning Kit (Invitrogen), following the manufacturer s protocol. Quantitative PCR. The first-round PCR was performed in the same volumes, tubes, and PCR cycler as the first-round PCR for terminal restriction fragment length polymorphism (T-RFLP) analysis. The second-round PCR was performed in 20-µl volumes in Hard- Shell ThinWall 96-Well Microplates (MJ Research) by using Ultra Clear Caps (MJ Research) and the DNA Engine Option 2 System (MJ Research). The melting curve analysis performed after each PCR showed a clear single peak at 90 C corresponding to the 491-bp amoa fragment. The size of this fragment was verified by means of agarose gel electrophoresis. Anomalies (due to contamination, primer dimer, false priming, etc.) were not evident in the negative first-derivative plots, as indicated by the lack of additional peaks. However, to avoid any possible primer dimer interference, the temperature at which the fluorescence was read during each cycle was adjusted to 86 C, a temperature above the melting point of the primer dimers. The amount of initial template DNA was estimated by determining the threshold cycle (Ct), the number of PCR cycles required for the fluorescence to exceed a threshold value higher than the background fluorescence. We assumed a threshold value of 25 times the background fluorescence (which we defined as the mean fluorescence values of the first three to eight PCR cycles).

2 2. Rotthauwe, J., Witzel, K. & Liesack, W. (1997) Appl. Environ. Microbiol. 63,

3 PCR Efficiency Controls. A defined amount of plasmid DNA (puc-plasmid, Invitrogen) was mixed with the same volume of soil DNA that was used for the regular real-time PCR. An 200-bp-long region of the plasmid was then amplified by using the primers M13F and M13R under the same PCR conditions described for the amplification of the amoa target (Table 1). Because the amount of the control gene was constant in all samples, any difference in Ct values was a direct measure of the different amplification efficiencies of the soil samples. We corrected for differences in PCR efficiencies by subtracting the Ct of the spiked control gene from the Ct of the target gene for each soil sample, as described by Meijerinck et al. (1). All control PCRs were performed in triplicate. 1. Meijerink, J., Mandigers, C., van de Locht, L., Tonnissen, E., Goodsaid, F. & Raemaekers, J. (2001) J. Mol. Diagn. 3,

4 Table 1. Primer description and thermal profiles for PCR amplification of amoa Primer Round of nested Thermal Molecular Sequence (5'-3') pair* PCR profile analysis A189 (1) GGNGACTGGGACTTCTGG 94 C, 30 s C, 30 s amoa-2r 1 CCCCTCKGSAAAGCCTTCTTC 72 C, 45 s (2) 30 cycles T-RFLP amoa-1f 94 C, 60 s GGGGTTTCTACTGGTGGT (2) 55 C, 90 s 2 72 C, 90 s amoa-2r CCCCTCKGSAAAGCCTTCTTC 18 cycles A189 GGNGACTGGGACTTCTGG 94 C, 45 s 56 C, 90 s 1 amoa-2r CCCCTCKGSAAAGCCTTCTTC 72 C, 180 s 13 cycles amoa-1f GGGGTTTCTACTGGTGGT 94 C, 45 s 55 C, 30 s Real-time PCR amoa-2r CCCCTCKGSAAAGCCTTCTTC 2 (Real-time PCR) 72 C, 180 s Plate read at 83 C 30 cycles Melting curve *Primers in bold represent the 5'-primer. All PCR profiles started with an initial denaturation at 94 C for 3 min and ended with a final elongation step at 72 C for 10 min, prior to holding the temperature at 4 C. Annealing temperature was decreased 0.5 C after each cycle until it reached 52 C. Primer labeled with 5-carboxyfluorescein. Melting curve analysis: C, 0.2 C per read, 1-s hold, final elongation step at 72 C for 10 min, prior to holding the temperature at 10 C. Control reactions with the primers M13F (5'-GTAAAACGACGGCCAG-3') and M13R (5'-CAGGAAACAGCTATGAC-3') were performed with identical thermal profiles. 1. Holmes, A. J., Costello, A., Lidstrom, M. E. & Murrell, J. C. (1995) FEMS Microbiol. Lett. 132,

5 2. Rotthauwe, J., Witzel, K. & Liesack, W. (1997) Appl. Environ. Microbiol. 63,

6 Table 2. Split-plot analysis of variance for the proportion of ammonia-oxidizing bacteria clade JR1 in the Jasper Ridge Global Change Experiment Source df Sum of Mean F P squares square Main plot comparisons C H C H Error (plot{h C}) Subplot comparisons N ** N C N H ** N C H Error (N plot{h C}) W W N W C W H ** W H C Error (W plot{h C}) N W C N W H * N W H C Error (N W plot{h C}) Two missing data points (for plots 47 and 123) were deleted. Split-plot ANOVA was performed by using the general linear model (GLM) in the SAS statistical package (SAS Institute, Cary, NC). *, P < 0.05; **, P < 0.01; N, nitrogen; H, heat; W, water; C, CO 2.

7 Table 3. Split-plot analysis of variance for the abundance of amoa genes in the Jasper Ridge Global Change Experiment Source df Sum of Mean F P squares square Main plot comparisons C * H C H Error (plot{h C}) Subplot comparisons N N C N H N C H Error (N plot{h C}) W W N W C * W H **** W H C Error (W plot{h C}) N W C N W H N W H C Error (N W plot{h C}) Two missing data points (for plots 47 and 123) were deleted. Split-plot ANOVA was performed by using the general linear model (GLM) in the SAS statistical package (SAS Institute, Cary, NC). *, P < 0.05; ****, P < ; N, nitrogen; H, heat; W, water; C, CO 2.

HiPer Real-Time PCR Teaching Kit

HiPer Real-Time PCR Teaching Kit Product Code: HTBM032 Number of experiments that can be performed: 10 Duration of Experiment Protocol: 1.5 hours Storage Instructions: The kit is stable for 12 months from