We performed DNA preparation and NGS library construction according to our previous methods14 (link) with slight modifications as follows. The powdered samples were digested in 0.5 M EDTA (pH 8.0) for 2 h at 56 °C in a rotating hybridization oven, and the supernatant was removed by centrifugation. This decalcification step was repeated three times in total. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1), followed by extraction with an equal volume of chloroform. After centrifugation, the aqueous solution was removed and subsequently concentrated by centrifugation dialysis using an Amicon Ultra-15 30 kDa centrifugal filter (Merck Millipore) to a final volume of 200 μL. The DNA solution was purified with silica-based MiniElute spin columns (Qiagen) according to manufacturer's protocol. The obtained DNA was quantified using Quant-iT dsDNA HS assay kit (Thermo Fisher Scientific). Using the obtained DNA, we prepared single- and double-stranded libraries.
Amicon ultra 15 30 kda centrifugal filter
Manufactured by Merck Group
The Amicon Ultra-15 30 kDa centrifugal filter is a laboratory device used for sample concentration and buffer exchange. It features a 30 kDa molecular weight cut-off membrane that retains molecules larger than 30 kDa while allowing smaller molecules to pass through during centrifugation.
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2 protocols using amicon ultra 15 30 kda centrifugal filter
1
DNA Extraction and NGS Library Preparation
2
DNA Extraction from Shell Powder
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DNA was extracted from the shell powder based on the so-called "Y1" method described in (Yang et al. 1998; Gamba et al. 2014 Gamba et al. , 2016)) . In short, powder was incubated overnight at 37°C in 15 mL digestion buffer (0.45 mM EDTA, 0.5% N-laurylsarcosyl, 0.25 mg/mL proteinase K) under constant mixing. After centrifugation (2,000 RPM; 2 min), the supernatant was filtered through an Amicon Ultra-15 30kDa centrifugal filter unit (Merck Millipore) (3,000 RPM; 50 min). DNA was then purified using the MinElute PCR Purification Kit (QIAGEN) in a final elution volume of 60 µL EB buffer.
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