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Ies can also achieve long-distance dispersal in this setting.Materials and
Although samples on the same rock platform could potentially be Cetilistat msds somewhat related (e.g., siblings or parents), sampling was carried out using the same criteria within each buy Octenidine (dihydrochloride) population so that, if diversity were underestimated due to relatedness of samples, this artefact should have been consistent across all sites. Each sample waswrapped in a fragment of CHUX?Superwipes? and multiple samples from each site were then placed in 50 ml vials containing 96 ethanol. Examination of individuals from each clade (by G.C. Zuccarello) confirmed their identities as B. intricata according to morphological criteria. For Adenocystis, one thallus was taken from each individual group of thalli, where ‘groups‘ were separated by at least 30 cm; each thallus was squeezed to remove as much internal moisture as possible, and multiple thalli were then placed in 50 ml vials containing 96 ethanol (up to 10 samples per vial). In the laboratory, samples were dried at 60 for between two and five hours. Desiccated voucher tissue from each sample has been kept in the laboratory, preserved with silica gel. For Bostrychia, sampling involved taking small (< 0.5 cm2) pinches of macroalgal tuft from separate clumps on the rock platform. Each sample waswrapped in a fragment of CHUX?Superwipes? and multiple samples from each site were then placed in 50 ml vials containing 96 ethanol. Examination of individuals from each clade (by G.C. Zuccarello) confirmed their identities as B. intricata according to morphological criteria. For Adenocystis, one thallus was taken from each individual group of thalli, where ‘groups‘ were separated by at least 30 cm; each thallus was squeezed to remove as much internal moisture as possible, and multiple thalli were then placed in 50 ml vials containing 96 ethanol (up to 10 samples per vial). In the laboratory, samples were dried at 60 for between two and five hours. Desiccated voucher tissue from each sample has been kept in the laboratory, preserved with silica gel. A small (< 1 mm2) fragment of tissue from each sample was isolated using flame-sterilised forceps. DNA extraction followed standard Chelex?procedure (Walsh et al., 1991). Extracted Adenocystis DNA was diluted one hundredfold before PCR; this step was not necessary for Bostrychia. For each species, a region of mtDNA (partial COI), cpDNA (partial rbcL; although in the case of Adenocystis the fragment amplified included only the last 198 bases of rbcL, followed by 224 bases of intergenic region), and rRNA (partial LSU). PCR amplification was carried out in 20 volumes containing 1.0 DNA, 0.5 of each primer, 1 x buffer, 0.8 mM dNTP, 1.5 mM MgCl2, and 1 U Taq polymerase (Bioline `BIOTAQ,‘ London, UK). Early trials with some relatively `universal‘ algal primers generally failed or yielded sequences contaminated by non-target DNA, perhaps from epiphytic material; specific primers were therefore developed for each marker for each genus (Table S2), with two exceptions for Adenocystis: the primers T01N(F) [24] and T13(R) [25] which successfully amplified a fragment of LSU, and cox 1-789F and cox 1-1378R [26] which sometimes successfully amplified a fragment of COI. Amplification was performed in an Eppendorf Mastercycler?ep gradient using the following profile: 94 for 2 min; forty cycles of 15s at 94 , 30s at 50 , 1 min at 72 , followed by a final 4 min extension at 72 .
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