Madison edited 16S rDNA Sequencing and Analysis (Organism Identification).md  over 9 years ago

Commit id: 6018c0cc3ab68d55475f6935c295d54f7ba918d3

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##Direct PCR (if not extracting DNA)  Centrifuge 1 ml of the overnight culture until the cells form a pellet at the bottom of the tube (about 5 minutes at 10,000 g), pour off the liquid on top (a.k.a. the supernatant) and resuspend in 100\(\mu\)l of sterile DNAase-free water. Incubate the samples at 100 °C deg C  for 10 minutes to help lyse the cells. Use the resulting solution as the template in the PCR reaction below. ##PCR reaction  This reaction uses the 27F (AGAGTTTGATCMTGGCTCAG) and 1391R (GACGGGCGGTGTGTRCA) primers which amplify a near full-length bacterial (and many archaeal) 16S rRNA gene. Our lab uses standard PCR reagents (Qiagen or Kappa), with an annealing temperature of 54 deg C and an extension at 72 deg C of 90 seconds. Do not forget to include positive (any sample containing bacterial genomic DNA) and negative (e.g., just water) controls.