Large-scale screens of metagenomic libraries.
Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.
Pham
Vinh D
VD
Division of Biological Engineering, Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, USA.
Palden
Tsultrim
T
DeLong
Edward F
EF
D001331:Automation; D005827:Genetics, Microbial / Q000379:methods*; D015698:Genomic Library; D009693:Nucleic Acid Hybridization; D012371:Robotics