DNeasy PowerFood Microbial Kit

Protocol tips

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	Solution PF1 must be warmed at 55°C for 5-10 minutes to dissolve precipitates prior to use. Solution PF1 should be used while still warm.

Protocol tips
Solution PF1 must be warmed at 55°C for 5-10 minutes to dissolve precipitates prior to use. Solution PF1 should be used while still warm.

Publication protocol

DNA extraction methods
Seven DNA extraction methods were evaluated to compare their relative efficiency with respect to the extraction of DNA from milk and cheese samples. The characteristics of the DNA extraction methods are summarized in Table 1. Methods 1–3 relied on the QIAamp® DNA stool mini kit (Qiagen Ltd, Crawley, West Sussex, UK), Chemagic Food Basic kit (Chemagen Biopolymer‐Technologie AG, Baesweiler, Germany) and Wizard® Magnetic DNA purification system for Food (Promega Corporation, Madison, WI, USA) to extract DNA from 200 mg of cheese or a pellet from 1 ml milk. Extractions were carried out according to the manufacturers’ instructions including a recommended modification to the QIAamp® protocol designed to enhance its ability to extract DNA from food matrices. Methods 4–5 employed the Milk Bacterial DNA Isolation kit (recommended by manufacturer’s for extracting DNA from milk) (Norgen Biotek Corporation, Ontario, Canada), and the PowerFood™ Microbial DNA Isolation kit (MoBio Laboratories Inc., Carlsbad, CA, USA), to extract DNA, again from a pellet obtained from 1 ml of milk or 1 ml of cheese homogenate (prepared by stomaching 1 g cheese with 9 ml tri‐sodium citrate), according to manufacturers’ instructions. Method 6 was designated the ‘Lytic’ method and represents a combination of methods used by O’Mahony and Hill (2004), Parayre et al. (2007) and Dolci et al. (2008). Here, DNA was isolated by resuspending the pellet (obtained from 1 ml milk or 1 ml of homogenized cheese) in 500 μl of breaking buffer for enzymatic lysis [20 mmol l−1 Tris HCl (pH8), 2 mmol l−1 EDTA, 2% Triton X100, 50 μg ml−1 lysozyme, 100 U mutanolysin] and incubated at 37°C for 1 h. Protein digestion was then performed by adding 250 μg ml−1 proteinase K and incubating at 55°C for 1 h. The suspension was transferred to a 2‐ml tube containing 0·3 g zirconium beads, the tube was shaken for 90 s in a bead beater, twice, and centrifuged at 12 000 g × 10 min. The supernatant was transferred to a fresh tube and combined with an equal volume of phenol : chloroform : isoamylalcohol (25 : 24 : 1) mixed gently and centrifuged at 12 000 g × 2 min. The top aqueous phase was transferred to a clean tube, and one‐tenth the volume 3 mol l−1 sodium acetate and 2 volumes of 100% ice‐cold ethanol were added. The suspension was mixed gently and stored at −20°C overnight. The sample was centrifuged at 14 000 g × 10 min, the supernatant removed and the pellet was washed with 70% ice‐cold ethanol followed by centrifugation at 12 000 g × 5 min and the pellet dried. The pellet was re‐suspended in 100 μl TE buffer. Finally, a ‘guanidine thiocyanate’‐based method was used, as described by Duthoit et al. 2003.

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Manufacturer protocol

Download the product protocol from Qiagen for DNeasy PowerFood Microbial Kit below.

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