A 3-mL volume of lysis buffer (20 mM Tris-HCl pH 8.0, 2 mM Sodium EDTA 1.2% Triton X-100) was added to 0.5 grams of stool sample, and the sample vortexed until homogenized. A 1.2 mL volume of homogenized sample and 15 μL of Proteinase K (Sigma Aldrich, PN. P2308) enzyme was aliquoted to a 1.5 mL tube with garnet beads (Mo Bio PN. 12830-50-BT). Bead tubes were then incubated at 65°C for 10 minutes and then 95°C for 15 minutes. Tubes were then placed in a Vortex Genie 2 to perform bead beating for 15 minutes and the sample subsequently spun in an Eppendorf Centrifuge 5424. 800 μL of supernatant was then transferred to a deep well block and DNA extracted and purified using a Chemagic MSM I (Perkin Elmer) following the manufacturer’s protocol. Zymo Onestep Inhibitor Removal kit was then performed following manufacturer’s instructions (Zymo Research PN. D6035). DNA samples were then quantified using Quant-iT on an Eppendorf AF2200 plate reader.
Onestep inhibitor removal kit
Manufactured by Zymo Research
The Onestep Inhibitor Removal kit is a tool used to purify nucleic acid samples by removing inhibitory substances that may interfere with downstream applications. The kit utilizes a simple, one-step process to effectively remove inhibitors from DNA or RNA samples, allowing for improved performance in various molecular biology techniques.
Automatically generated - may contain errors
2 protocols using onestep inhibitor removal kit
1
Stool DNA Extraction and Purification
2
Automated DNA Extraction and Purification
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Bead tubes were then incubated at 65 °C for 10 minutes and then 95 °C for 10 minutes. Tubes were then placed in a Vortex Genie 2 to perform bead beating for 13 minutes and the sample subsequently spun in an Eppendorf Centrifuge 5424. Supernatant (700 μL) was then transferred to a deep well block. DNA extraction and purification was performed using a Chemagic MSM I (Perkin Elmer) following the manufacturer’s protocol. Samples were then further purified using the Onestep Inhibitor Removal kit following manufacturer’s instructions (Zymo Research PN. D6035). DNA samples were then quantified using Quant-iT on an Eppendorf AF2200 plate reader.
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