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Following the second dilution streaking, the organisms need to be identified. 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 a method originally developed by Fred Sanger and now known as "Sanger sequencing". There are multiple approaches one can take to these steps. For example, the PCR reaction needs DNA from the organisms of interest. That DNA can come directly from a liquid culture of the organism (when this is used for PCR this is known as direct PCR). Alternatively, one can take a liquid culture and then isolate the DNA from that culture and use the "clean" DNA as material for the PCR. This adds an extra step to the process - a step known as "DNA extraction". Direct 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 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 be used  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 Purification Kit. Follow the protocol or kit instructions provided by the manufacturer and then proceed to "PCR reaction" below.