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First-round PCR products (5?��l) were run on 1% agarose gels and quantified using known concentrations of a Hyperladder (Bioline, London, UK). Agarose gels were imaged using a gel documentation system, and individual bands were quantified with the GelEval software (Frogdance Software, http://www.frogdance.dundee.ac.uk) by comparing pixel intensities of samples to reference bands from the Hyperladder. Lower band intensities were regarded as a rough estimate for lower template concentrations for PCR in the samples (i.e., lower standing stock or active prokaryotes based on DNA and RNA profiles, respectively). Amplification products were analysed using DGGE (as partially shown in Fig. 3) as described previously (Muyzer et al., 1993) using 8% polyacrylamide gels, with a denaturant gradient of 35 to 65%, run for 16?h at 75?V and 60?��C. Sensitivity of band detection was increased [http://www.selleckchem.com/products/Rapamycin.html Rapamycin supplier] by silver staining (Mahmood et al., 2005). Bands were considered to represent OTUs and differences in banding patterns indicated differences in OTU composition. Separate binary matrices were constructed for bacterial and crenarchaeal DGGE gels, based on the presence or absence of OTUs and results were used to determine OTUs that were present in all [http://en.wikipedia.org/wiki/IRS1 IRS1] samples from a particular sediment horizon and those shared between DNA- and RNA-targeted analyses in samples taken at all sampling times (June, September and February). The binary matrix was loaded into Primer6 (Primer-E, Lutton, UK) and cluster analysis was used as previously described to evaluate the significance groupings (Bertics and Ziebis, 2009). Plainstat (Plainstat Software, http://www.plainstat.com) was used to perform Student��s t-tests [http://www.selleckchem.com/products/jq1.html selleck chemical] of significance. Significance of clustering of DGGE profiles was determined using SIMPROF in the Primer6 package ( Bertics and Ziebis, 2009). DNA concentration of extractable DNA was significantly higher in all samples collected in February than June and September (Fig. 2a). Furthermore, DNA concentration in June and September at sediment horizons 2�C5?cm was significantly greater (p