As my lab strives to adopt increasingly high throughput molecular practices, we've hit a few bumps along the way. One involves the first step in the process of obtaining DNA sequence data: extraction. As a molecular biologist, I've grown up in the era of kits: I've always done genomic extractions by pulling a Qiagen/Promega/Viogene kit off the shelf, dropping a chunk of tissue into a tube and following instructions. This simple and convenient approach, however, is challenged by the needs of the very high throughput facility we're working with. They're asking for genomic DNA with a 260/280 ratio (a measure of a sample's quality that reflects the ratio of DNA to undesirable proteins) between 1.8 and 2.0. Essentially, they're asking for genomic DNA that is completely free of protein contaminants. Unfortunately, kits that rely on a silica filters tend to give us ratios in the 1.2-1.3 range. After a week of experimentation, we've been able to get samples with ratios in the 1.6-1.7 range, but only via phenol-chloroform extraction. Although I've spent my career avoiding a method that has always been described as "unpleasant", several gel-based methods make the phenol-chloroform process relatively painless (check out Phase Lock Gel from Eppendorf [apparently discontinued] and MaXtract from Qiagen). I'm sure we're not the first to encounter this problem, but hopefully this post will help others avoid the trouble of extracting dozens of samples only to be told later that the resulting samples weren't good enough!
Dechronization is authored by evolutionary biologists interested in the development and application of methods for estimating phylogeny and making phylogeny-based inferences. The goal of the blog is to provide a forum for discussion of the latest research and methods, while also providing anecdotes, tidbits of natural history, and other related information.