Jonathan A. Eisen edited 16S rDNA Sequencing and Analysis (Organism Identification).md  almost 10 years ago

Commit id: 17d8ec29cba6f045ad81af680d3c70fb5943d89f

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##Direct PCR (if not extracting DNA)  Centrifuge 1mL 1 mL  of the overnight culture until the bacteria cells  form a pellet at the bottom of the tube (about 5 minutes at 10,000g), 10,000 g),  pour off the supernatant liquid on top (a.k.a. the supernatant)  and resuspend in 100\(\mu\)l of sterile DNAase-free water. Incubate the samples at \(100\,^{\circ}\mathrm{C}\) for 10 minutes to help lyse the cells. Use the resulting solution as the template in the PCR reaction below below.  ##PCR reaction  This reaction uses the 27F (AGAGTTTGATCMTGGCTCAG) and 1391R (GACGGGCGGTGTGTRCA) primers which amplify a near full-length bacterial 16S rRNA gene. (NOTE - SHOULD SAY SOMETHING HERE ABOUT ARCHAEA AND WHETHER THESE PRIMERS WILL WORK FOR THEM).  Our lab uses standard PCR reagents (Qiagen or Kappa), with an annealing temperature of \(54\,^{\circ}\mathrm{C}\) and an extension at \(72\,^{\circ}\mathrm{C}\) of 90 seconds. Don't Do not  forget to include positive (any sample containing bacterial genomic DNA) and negative (water) (e.g., just water)  controls. After PCR is completed, confirm the PCR reaction worked by agarose gel electrophoresis, all controls behaved as expected, and that you have DNA fragments of the correct size (~1350bp).