Samples are from the SRE4 experiment - an in situ experiment to determine fate and effects of different types of oils in the Antarctic marine environment. For details see: Powell S.M., Snape I., Bowman J.P., Thompson B.A.W., Stark J.S., McCammon S.A., Riddle M.J. 2005. A comparison of the short term effects of diesel fuel and lubricant oils on Antarctic benthic microbial communities. Journal ... of Experimental Marine Biology and Ecology 322:53-65.
Samples were analysed by denaturing gradient gel electrophoresis (DGGE) with primers specific for the Desulfobacula group.
Samples A,B,C,D,E,F,G,H,I are all initial samples collected different days
Samples beginning T0 are predeployment samples, the next number refers to the batch.
Samples beginning T2 are 1 year samples with:
C = control
S = SAB
L = lubricant
U = used lubricant
B = biodegradable lubricant
PCR conditions were as follows:
Primers: 764F: ACAATGGTAAATGAGGGCA
1392RC: CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCCACGGGCGG TGTGTAC
50 ul (micro litre) reactions with Advantage II taq (Clontech) following manufacturer's recommendations with 20 pmol (pico mol) each primer and 20 ng (nano gram) template DNA.
Cycling: 94C 5 minutes
10 cycles of 94C 1 minutes
65C 1 minutes (-1C per cycle)
72C 2 minutes
20 cycles of 94C 1 minutes
55C 1 minutes
72C 2 minutes
72C 30 minutes
DGGE carried out using the D-Code system (BioRad).
Gel: 8% acrylamide 30 - 65% denaturant with 2 cm stacking gel (15% acrylamide)
1 x TAE, 60 degrees C, 70V 16 hours
The gels were pre-run for 20 minutes then half reaction volume was loaded and the lanes flushed out after 15 minutes.
Gels were stained with SYBRGold.
Images were captured using Storm Phosphorimager and ImageQuant v5.2 software(.gel files).
Samples were only compared within a gel. Band pattern results are in the file desulfodgge.xls. For each comparison made there is a separate sheet in this file (see below). The first column in each sheet is the band position (or band name) and the remaining columns are samples with the first row being the sample name. '0' '1' indicate the band was 'absent' or 'present'.
Comparison Image files (.gel and .tif) results sheets
Background variation 140704f; 140704b 140704f and 140704b
predeployment batches 180604f; 180406b 180604f and 180604b
effect of setup 150704 150704
immediate effect of oil 250604f; 250604b 250604f and 250604b
1 year samples (T2) 040804f; 040804b 040804f and 040804b
This work was completed as part of ASAC projects 1228 and 2201 (ASAC_1228, ASAC_2201).