deletions | additions       diff --git a/16S rDNA Sequencing and Analysis (Organism Identification).md b/16S rDNA Sequencing and Analysis (Organism Identification).md  index 2a0661a..1d520b7 100644  --- a/16S rDNA Sequencing and Analysis (Organism Identification).md  +++ b/16S rDNA Sequencing and Analysis (Organism Identification).md 
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The samples can be prepared for 16S rDNA PCR via direct PCR (using the liquid culture as the template for the PCR reaction) or following DNA extraction. Direct PCR significantly decreases the amount of work needed for preparation, but it can yield poorer Sanger results, both in terms of PCR success and resultant sequence quality. However, we recommend direct PCR when screening a large number of samples. DNA extraction can then be used for any recalcitrant samples. DNA extraction is significantly more work, but it often generates better Sanger sequences allowing for more accurate identification.  ##DNA Extraction  There are a number of different options for DNA isolation and which one should use depends on many factors including available equipment, experience, and cost. A standard approach in microbiology involves the use of phenol and chloroform extraction followed by ethanol precipitation, and any number of protocols for this approach can be found in books, articles and on the internet. A common alternative approach is to use a commercially available kit - there are many advantages to such kits - notably ease and lack of toxic chemicals. A disadvantage of kits is that they typically are more expensive per sample than other approaches (especially if one is only doing a few samples since most kits include materials for at a minimum 50 samples). For most projects, we use kits - especially the Promega-Wizard Genomic DNA PurificationKit or the MO BIO-Power Soil DNA Isolation  Kit. Follow the protocol or kit instructions provided by the manufacturer and then proceed to "PCR reaction" below.  ##Direct PCR (if not extracting DNA)  Centrifuge 1 mL 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 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.