Magnetic bead-based solution for DNA extraction from environmental samples using Mag-Bind® Environmental DNA 96 Kit from Omega Bio-tek

Kiranmai Durvasula¹, Julie Baggs¹, Jessie Chen¹, Travis Butts¹

¹Omega Bio-tek, Inc., Norcross, GA 30071

Introduction

The study of environmental metagenomics is key in unraveling the complex diversity of the microorganisms that inhabit different ecosystems and their contribution to the key characteristics of these ecosystems. The outcome of these studies has several different fields. Some of the applications include water quality assessments, agricultural microbiome analysis studies to improve soil quality, crop production, ecological remediation, and other biological investigations on environmental samples etc. Nucleic acid extraction is the first crucial step and sufficient amounts of high-quality nucleic acids representative of all the organisms present in a given environmental sample must be obtained for any comprehensive downstream analysis. Environmental samples are particularly challenging owing to the presence of naturally occurring inhibitors such as humic acid, fulvic acid, and other organic matter that can co-purify along with the DNA potentially interfering with sensitive downstream applications. Omega Bio-tek’s Mag-Bind® Environmental DNA 96 Kit (M5645) was developed to isolate high quality DNA from various environmental samples such as soil, water, etc. and uses a proprietary inhibitor removal agent (cHTR) to eliminate inhibitors from the extraction process. The kit uses magnetic beads for purification, making it amenable for high-throughput processing on various open-ended liquid handlers or magnetic processors. We validated the Mag-Bind® Environmental DNA 96 Kit on soil and water samples and compared its performance with Company Q’s soil and water DNA isolation kits, respectively. The kit performance was evaluated in terms of yield and quality of the DNA obtained, as well as sensitivity of real-time PCR amplification downstream.

Materials and Methods

Soil DNA Extraction

Outdoor soil (250 mg) was spiked with approximately 1 mL culture of Bacillus subtilis and DNA was extracted using Mag-Bind® Environmental DNA 96 Kit (Omega Bio-tek) and Soil DNA Isolation kit (Company Q) following manufacturer’s recommended protocols. The isolations were carried out in quadruplicate for the Omega kit and in duplicate for the competitor’s kit.

Water DNA Extraction

Pond water (100 mL) was spiked with approximately 1 mL culture each of B. subtilis and E. coli harboring pGEM plasmid and was filtered through Sterivex (Millipore SVGPL10RC) filters. Upon filtration, DNA was extracted from these filters in quadruplicate following manufacturer’s recommended protocols.

DNA Quantification and Quality Assessment

Following the soil and water DNA extraction protocols, purified DNA was eluted in 100 µL elution buffer included in each kit and quantified using Thermo Scientific’s NanoDrop® 2000c. The quality of the purified DNA was analyzed by performing real-time PCR using B. subtilis specific primers on undiluted and 10-fold diluted DNA isolated from soil extraction protocol and on 10-fold and 100-fold diluted DNA isolated from water extraction protocol. Briefly, a qPCR reaction was set up to a total volume of 20 µL using Agilent’s Brilliant III 2X SYBR® as the master mix and 2 µL of template DNA at appropriate dilutions amplified with suitable primers following a standard protocol on the ABI 7900.

Results and Discussion

The DNA yields and absorbance ratios (A260/A280 and A260/A230) from the soil samples spiked with B. subtilis cells using the Omega kit and Company Q’s kit are as shown in Table 1. The average DNA yield from the Omega kit was ~3.03 µg compared to 1.4 µg from Company Q’s kit corresponding to a 116% increase in yield using the Omega kit. The average A260/A280 and A260/A230 ratios using Omega Bio-tek’s kit were better compared to Company Q’s. The A260/A280 ratios were 1.86 ± 0.03 (Omega) and 1.37± 0.05 (Company Q) and A260/A230 ratios were 1.13 ± 0.07 (Omega) and 0.65 ± 0.05 (Company Q) (Table 1). These ratios suggest superior DNA quality post extraction using Omega Bio-tek’s kit when compared to Company Q’s kit.