Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

Summary

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

Abstract

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications.

The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (β-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

Protocol

Part I: Extraction of DNA Procedure 1. 8 hours are required, for processing 6 samples excluding quantification of DNA. Before you start the extraction, you will need to pre-chill mortar, pestle and spatula in a -20 °C freezer or using liquid nitrogen. Also, pre-chill 50 ml tubes containing 20 ml chloroform isoamyl alcohol (24:1) on ice. Set the hybridization oven to 65 °C to pre-heat. Check CTAB solution, if it is…

Materials

Recipe

* Denaturing solution
4 M Guanidine isothiocyanate
10 mM Tris-HCl (pH 7.0)
1 mM EDTA
0.5% 2-mercaptoethanol

Denaturing solution: 10 ml in total
Guanidine isothiocyanate (MW 118.16)	4.73 g
1M Tris-HCl (pH 7.0)	100 μl
0.5M EDTA	20 μl
Add water to 9.95 ml in total.
Autoclave.
Add 50 μl 2-mercaptoethanol just before use. (5 μl of 2-mercaptoethanol per 1 ml Denaturing Solution) Note: Keep the Denaturing solution at 4 °C.  Do not use buffer older than one week. If possible, make fresh buffer to use.

**Extraction Buffer
100 mM Sodium phosphate buffer [pH 7.0]
100 mM Tris-HCl [pH 7.0]
100 mM EDTA [pH 8.0]
1.5 M  NaCl
1% Hexadecyltrimethylammonium bromide (CTAB)
2% SDS

Extraction Buffer for 1 L in total
1M Sodium phosphate buffer [pH 7.0]*	100 ml
1M Tris-HCl [pH 7.0]	100 ml
0.5M EDTA [pH 8.0]	200 ml
5 M NaCl	300 ml
Autoclave and keep it at room temperature.
Add 200ml, 5% Hexadecyltrimethylammonium bromide (CTAB, autoclaved) and 100 ml, 20% SDS (autoclaved) just before use. If CTAB was crystallized, melt it at 60 °C. * 1M Sodium phosphate buffer: 57.7 ml, 1M Sodium phosphate monobasic (NaH2PO4) and 42.3 ml, 1M Sodium phosphate dibasic (NaH2PO4). Adjust pH to 7.0 Note: Do not use 20 % SDS if it has precipitation. It is normal to see milky suspension when you add SDS to the solution. Once you add SDS, place the extraction buffer at 60 °C to ensure SDS is well suspended.