* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Supplementary Information (doc 36K)
Survey
Document related concepts
Comparative genomic hybridization wikipedia , lookup
Metagenomics wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
DNA vaccination wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
DNA supercoil wikipedia , lookup
Molecular cloning wikipedia , lookup
Transformation (genetics) wikipedia , lookup
History of genetic engineering wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
Transcript
Disconnect of microbial structure and function: enzyme activities and bacterial communities in nascent stream corridors Frossard Aline, Linda Gerull, Michael Mutz and Mark O. Gessner Supplementary Methods: Detailed information on DNA extraction and PCR reactions DNA extraction and purification involved cell breakage, enzymatic digestion of unwanted cell constituent, and DNA purification. Frozen soil and sediment samples (0.5 g fresh mass) were placed in sterile 2-ml reaction tubes containing 0.5 g zirconium beads (0.7 mm diameter, Carl Roth GmbH + Co. KG, Karlsruhe, Germany). Cells were mechanically disrupted by shaking the tubes on a microplate shaker (IKA, VWR, Dietikon, Switzerland). A volume of 600 μl phosphate buffer (53 ml l-1 of 120 mM NaH2PO4 and 947 ml l-1 of 120 mM Na2HPO4, pH=8) and 100 μl of 25% sodium dodecyl sulfate (SDS) was then added. The tubes were fixed horizontally on a shaker, mixed for 10 min, and finally centrifuged for 6 min at 15230 × g. The supernatants were transferred to new 2-ml reaction tubes. Enzymatic digestion of the cells was achieved by adding to the reaction tubes 200 μl of lysozyme in TE buffer (10 mg/ml, TE buffer: 10 mM Tris, 1 mM Na2EDTA, pH=8) and placing them on a thermomixer (37°C) for 30 min. Next, 12.5 μl of proteinase K (20 mg/ml) and 150 μl of SDS 25% was added before continuing incubation of the tubes at 55°C overnight. Finally, the DNA was purified and proteins removed from the solution by adding 7.5 M ammonium acetate (0.4x the final volume), placing the tubes on ice for 5 min, and centrifugation for 8 min at 15230 x g. The supernatant was transferred to a new reaction tube and a volume of isopropanol corresponding to 70% of the volume of the supernatant was added. The tubes were then centrifuged for 60 min at 15230 × g and the supernatant discarded. The pellet was washed twice by shaking the tube with the pellet and with 600 μl of 80% ethanol for 15 s, centrifuging for 5 min at maximum speed (17135 × g), and discarding the supernatant each time. The pellet in the tube was then dried for 1-2 hours in a clean bench and finally re-dissolved in 100 μl of Nanopure water (4°C, overnight). The extracted and purified DNA was stored at -20°C. PCR amplification of the V3 region of 16S rDNA gene was performed in two steps to obtain enough DNA for the analyses. A first PCR was carried out using forward primer Eub338f (5’-ACT CCT ACG GGA GGC AGC AG-3’) and reverse primer Eub518r (5’-ATT ACC GCG GCT GCT GG-3’; Microsynth, Balgach, Switzerland). The same universal bacterial primers were used for the second PCR except that a 40 bp GC-clamp needed for denaturating gradient gel electrophoresis (DGGE) analysis was added to the 3’ end of the forward primer. The PCR reaction mix contained (final concentrations): 1x GoTaq® Flexi reaction buffer (Promega, Switzerland), 3 mM MgCl2, 0.25 mM dNTPs, 0.25 mM of each primer, and 1 U/μl GoTaq® Flexi DNA Polymerase (Promega, Dübendorf, Switzerland). DNA extract (2 μl) was added to each reaction mix and the final reaction volume adjusted to 20 μl. A first denaturation step was performed at 94°C for 5 min. Each of the following 31 amplification cycles consisted of a heat denaturation step at 94°C for 1 min, primer annealing at 65°C for 30 s and an extension phase at 74°C for 1 min. During primer annealing, a touchdown of 1°C per cycle was programmed for the first 10 cycles. The mix was then maintained at 74°C for 10 min for the final extension. The size and yield of the PCR products was checked on a 2% agarose gel containing a 100 bp ladder (Promega, Dübendorf, Switzerland).