IMIS - Marine Research Groups

Two parallel cores were sampled using Kovacs Mark II 9 cm corer (Kovacs Enterprise, Roseburg, Oregon) and a 36 V electric hand drill. Prior to drilling, the core barrel was rinsed with sterile deionized water (Milli-Q Integral Water Purification System, EMD Millipore Corporation, Billerica, MA). The two cores were immediately sectioned on site to 30-cm intervals using a hand saw (rinsed in deionized water and wiped with an ethanol wipe) and placed in UV-sterilized polypropylene bags.

Study Extent: Two ice cores were taken during May 2011 at one site on landfast ice off the northern shore of northern Ellesmere Island Nunavut, Canada (82.54905°N, −62.37685°W).

Method step description:

1. Ice samples used were thawed at room temperature in the dark, and were filtered individually through 0.22-μm-pore-size polyethersulfone membrane filters (Pall, Mississauga, ON, Canada). Direct melting was used to avoid nonspecific addition of DNA and dilution of samples. All glassware was sterilized using 10% household bleach solution followed by thorough washing in sterile deionized water between samples to prevent cross-contamination. Each filter was placed in a microfuge tube and submerged in RNAlater® solution (Life Technologies, Burlington, ON, Canada) and stored at −20 °C for later DNA extraction.
2. DNA was extracted from preserved filters by bead beating using the FastDNA® SPIN Kit for Soil (MP Biomedicals, Solon, OH) as instructed by the manufacturer. Filters from equivalent 30-cm sections of duplicate ice cores were combined prior to DNA extraction to ensure sufficient DNA yield. Seawater duplicate samples were processed individually.
3. Molecular Research LP (Shallowater, TX) performed pyrosequencing of the V1-V3 regions of the bacterial 16S rRNA gene as described in Dowd et al. (2008). Amplification was performed using HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA) under the following conditions: 94 °C for 3 min, followed by 28 cycles of 94 °C for 30 s; 53 °C for 40 s; and 72 °C for 1 min with a final elongation step at 72 °C for 5 min. Gene-specific forward PCR primer sequences were tagged with the sequencing adapters for GS FLX Titanium chemistry, an 8 base barcode, and a linker sequence. All PCRs were performed in triplicate, mixed in equal concentrations, and purified using Agencourt AMPure beads (Agencourt Bioscience Corporation, MA). FLX-Titanium amplicon pyrosequencing was performed using the Genome Sequencer FLX System (Roche, Branford, CT).