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#16S rDNA Sanger Sequencing  Following liquid culturing, the organisms need to be identified, or classified. This is accomplished by determining and then analyzing the DNA sequence of the 16S rRNA gene. In this section, we describe how the sequence of this gene is determined and readied for analysis. The general outline is as follows: DNA extraction, polymerase chain reaction (PCR) amplification of the 16S rRNA gene, and sequencing of the resulting PCR product using Sanger sequencing \cite{Sanger_1977}. There are multiple approaches one can take to these steps. For example, the PCR requires DNA from the organisms organism  of interest. That DNA can come directly from a liquid culture of the organism (when this is used for PCR this is known as colony PCR). Alternatively, one can take a liquid culture and then isolate the DNA from that culture and use the purified DNA as input  material for the PCR. This adds an extra step to the process - a step known as DNA extraction (see below.) Colony PCR significantly decreases the amount of work needed for preparation, but it can yield poorer results, both in terms of PCR success and resultant sequence quality. However, we recommend colony 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 extraction,  and which one should be used depends on many factors including available equipment, experience, and cost. A standard approach in microbiology involves the use of a 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 samples,  since most kits include materials for a minimum of 50 samples). For most projects, we use kits - typically the Promega-Wizard Genomic DNA Purification Kit. Follow the protocol or kit instructions provided by the manufacturer and then proceed to "PCR" below. 
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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 (the supernatant) and resuspend the pellet in 100 μl of sterile DNAase-free water. Incubate the samples at 100°C for 10 minutes to help lyse the cells. Use the resulting solution as the template in the PCR below.  ##PCR  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 °C 54°C  and an extension at 72 °C 72°C  of 90 seconds. Do not forget to include positive (any sample containing bacterial genomic DNA that you have successfully amplified before) and negative (_e.g_., replace DNA with water) controls. The full program we use is: 1: 95 °C 95°C  for 2:00 2: 95 °C 95°C  for :15 0:15  3: 54 °C 54°C  for :30 0:30  4: 72 °C 72°C  for 1:30 5: Go to 2 (40 times)  6: 72 °C 72°C  for 3:00 7: 4 °C 4°C  forever After PCR is completed, confirm the PCR worked by agarose gel electrophoresis, all controls behaved as expected (i.e. (_i.e._,  band in the positive control and no band in the negative control), and that you have DNA fragments of the correct size (~1350bp). ##Submit Samples for Sequencing  Very few single-researcher labs currently have the capacity to do Sanger sequencing. However, there are a number of DNA sequencing facilities (commercial and academic) that provide Sanger sequencing services for researchers. They will handle as little as a single sample, or will allow you to submit an unlimited number of samples,typically  arrayed in 96-well plates. You will typically provide both your PCR product as well as primers for sequencing (typically, the (the  same primers used for PCR are usually  used for sequencing). To get the most data, do not forget to request both  forward (_e.g_., using primer 27F) and reverse (_e.g._, using primer 1391R) reactions for each sample. Each facility will have its own guidelines concerning DNA and primer concentration. Our lab uses the [UC Davis Sequencing Facility](http://dnaseq.ucdavis.edu). If an internet search does not reveal the presence of a Sequencing Facility near you, most sequencing centers will allow you to ship samples to them for sequencing. Another possibility is [Science Exchange](https://www.scienceexchange.com/) which is an online clearinghouse for lab services.