Denaturing Gradient Gel Electrophoresis in Marine Microbial Ecology

Koninklijk Nederlands Instituut voor Onderzoek der Zee - NIOZ, Burg, North Holland, Netherlands
Methods in Microbiology (Impact Factor: 0.08). 01/2001; 30:425-468. DOI: 10.1016/S0580-9517(01)30057-0
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Article: Denaturing Gradient Gel Electrophoresis in Marine Microbial Ecology

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    • "Total DNA was obtained directly from cells trapped in the filters using the methods described in Liu et al. (2013). The bacterial 16S rRNA genes were amplified with primers 341F- GC (5′-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GCC TAC GGG AGG CAG CAG-3′) and 907R (5′-CCG TCA ATT CMT TTG AGT TT−3′), which are specific for most bacteria (Schäfer and Muyzer 2001). For the microeukaryotic species, PCR primer pair Euk1A (5′-CTG GTT GAT CCT GCC AG-3′) and Euk516R-GC (5′-ACC AGA CTT GCC CTC CCG CCC GGG GCG CGC CCC GGG CGG GGC GGG GGC ACG GGG GG−3′) were used for amplification of 18S rRNA genes (Díez et al. 2001). "

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    • "The resulting DNA samples were then subsequently stored at −20°C until further analysis. PCR Amplification of the 16 S rRNA V3 Region from the Biofilm Microbes From the extracted biofilm DNA, the V3 region of the 16S bacterial rRNA gene was amplified through polymerase chain reaction (PCR) according to previously-described conditions (Schafer and Muyzer 2001). Briefly, 40-μL reactions were prepared consisting of 1 × iTaq amplification buffer [Bio-Rad Laboratories (Canada), Mississauga, Ontario], 200 nM of each dNTP, 500 nM of each primer, 2 mg ml −1 BSA, and 2U iTaq polymerase [Bio-Rad Laboratories (Canada) Mississauga, Ontario]. "
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    • "V3–V5 16S rRNA gene tag sequencing Variable regions 3 to 5 (V4–V5) of the 16S rRNA gene from Bacteria and Archaea were amplified from a selected number of unamended slurry DNA samples covering a range of temperatures (25, 35, 38, 46, 66 • C) and time points (0, 15, 35, 62 days) using barcoded fusion primers 357F/907R (Muyzer, Dewaal and Uitterlinden 1993; Muyzer et al. 1998 "
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