Sample Preparation |
Purification of Genomic DNA
Order
| Protocol
| Related Products
Direct Isolation of Metagenomic DNA from Environmental Water Samples - 715k PDF
Poster from ASM 2008 -
270k PDF
Applications
- Easy isolation of fosmid cloning-ready metagenomic DNA from microbes, including
Gram-positive bacteria, in a water sample.
- Isolation of metagenomic DNA for PCR or next-gen sequencing.
The Metagenomic DNA Isolation Kit for Water was developed to isolate metagenomic
DNA directly from culturable or unculturable microbes in a water sample, to
generate cloning-ready DNA for construction of random-sheared fosmid libraries.
Traditional methods for isolation of metagenomic DNA to be used in fosmid library
construction involve agarose plugs, random shearing, and a size selection using
pulse-field gel electrophoresis before cloning into a fosmid vector. These
methods (Fig. 1B) are time-consuming, inefficient, and result in a library
with biased genome representation. The Metagenomic DNA Isolation Kit for Water
isolates high-molecular-weight DNA that is randomly sheared (with fragment
sizes approximately 40 kb), does not require size selection (Fig. 1A), and
can be used directly for end-repair and cloning into the EPICENTRE pCC1FOS™ Vector
(CopyControl™
Fosmid Library Production Kit, sold separately).
The kit protocol uses Ready-Lyse™ Lysozyme followed by Proteinase K
treatment to isolate high-molecular-weight DNA. Using this kit, DNA isolated
from 300 ml of lake water can result in a fosmid library containing as many
as 100,000 metagenomic clones.
Benefits
- No bead-beating.
- No agarose plugs, mechanical shearing, or size selection.
- Prevents loss of DNA, thereby generating unbiased libraries.
- No columns or phenol/chloroform extractions.
- Fosmid cloning-ready DNA (Fig. 2) in 90 minutes.
- Improved yield, increasing the probability of recovering DNA from
low-abundant organisms present in the collective genomes in environmental
samples.
|
Figure 2. Size of inserts from a metagenomic fosmid library. Metagenomic
DNA isolated from lake water was used to generate a fosmid library using
the CopyControl™ Fosmid Library Production Kit. DNA was isolated
from randomly selected clones and digested with Not I. The insert
sizes were approximately 40 kb. Lane M, Kilobase ladder; lane 1, 40-kb
control insert; lanes 2-19, Not I-digested fosmid DNA. |
|
| Figure 1 (click to enlarge). Comparison of traditional
and EPICENTRE procedures for metagenomic DNA isolation and library construction. |
CopyControl™ Fosmid Library Production Kit
CopyControl™ HTP Fosmid Library Production Kit
[top of page]
|
