Large-Scale Screens of Metagenomic Libraries

Abstract

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.

Protocol

1. Hybridization procedure

One hybridization tube will take 1 or 2 membranes (size 22 by 22 cm). Use 50 ml of hybridization mixture and 0.5 mg of DNA probe (10 ng/ml final concentration) per hybridization tube. For best results, use gel-purified DNA as probe. If scaling up probe preparation, increase number of tubes rather than increasing the amount of reagents per tube.

Probe preparation (0.5 mg DNA):

1. Dilute 15 μl of cross-linker solution with 60 μl of water.
2. Dilute DNA to 10 ng/μl with water to 50 μl total volume in a screw-capped microtube.
3. Denature DNA for 5 min in boilin....

Discussion

The stripping procedure has not been optimized. However, the stripping procedure given in the manufacturer's instruction (2 washes each, 10 min in 100% ethanol, 1 wash of 1 h at 60°C in 0.5% SDS) is insufficient. At least overnight incubation in 100% ethanol is necessary to dissolve the ECF reaction product; several days incubation in 100% ethanol appears to have no adverse effects on the ability to reprobe the membrane. The manufacturer's SDS treatment also appears to be insufficient. NUF has tried 1 h at 80°C w.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AlkPhos Direct Labelling Reagents	kit	Amersham	RPN3680	Contains labeling reagent, cross-linker solution, reaction buffer, water, control DNA, hybridization buffer, blocking reagent. Store hybridization buffer and blocking reagent at room temperature, other reagents at 2-8°C.
ECF Substrate	kit	Amersham	RPN3685	Contains ECF substrate, ECF substrate dilution buffer.Note: Pack contains total 60 ml reagent; aliquot into 10 ml samples in Falcon tube and store at 20°C. Use about 10 ml per membrane (size of 22 by 22 cm).
20x Secondary Wash Buffer	buffer			121 g Tris(final 1 M), 117 g NaCl(final 2 M).Adjust pH to 10. Adjust to 1 L with water. Store at 2-8°C for up to 4 months.
0.5 M NaH2PO4 buffer pH 7	buffer			69 g NaH2PO4.H2O. Adjust pH to 7 with NaOH. Adjust to 1 L with water. Store at room temperature.
1 M MgCl2				50.8 g MgCl2.6H2OAdjust to 250 ml with water. Store at room temperature.
10% (w/v) SDS				20 g SDSAdjust to 200 ml with water. Store at room temperatur
Combination pack RPN3692 = RPN3680 + RPN3685