Food DNA isolation miniprep kit: Manual

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Procedures Reminder • All steps are to be carried out at room temperature unless stated otherwise • Wash Buffer 1 and Wash Buffer 2 (concentrate) have to be diluted with absolute ethanol before use. Please refer to Reconstitution of Solutions. • If precipitation forms in Buffer FB, incubate at 55°C - 65°C with occasional mixing until precipitate is completely dissolved. Pre-set waterbath to 65°C. Pre-heat Elution Buffer at 65°C. 1. Sample preparation and cell lysis a. Solid Sample ♦ Grind the sample into powder form using commercial homogenizer or liquid nitrogen with pestle and mortal. Lysis is more efficient when the sample is well homogenized. Homogenization method used depends on the type of sample. In some cases, homogenization is not needed but complete suspension is necessary for efficient lysis for samples such as soy flour and cocoa powder ♦ Place 100-200mg of ground sample into a clean microcentrifuge tube. Add 700µl Buffer FL and 20µl Proteinase K into the sample. Mix thoroughly by vortexing to obtain a homogenous solution. Incubate at 65°C for 30 min with occasional mixing during incubation to ensure thorough lysis of the sample. Use up to 100mg for hygroscopic samples e.g. flour, dried plant, etc. Users may need to increase the volume of Buffer FL until the sample is semi-fluid like. b. Liquid sample ♦ Transfer 400µl of sample directly into a clean microcentrifuge tube. Add 300µl Buffer FL and 20µl Proteinase K into the sample. Mix thoroughly by vortexing to obtain a homogenous solution. Incubate at 65°C for 30 min with occasional mixing during incubation to ensure thorough lysis of the sample.
Optional: Removal of RNA If RNA-free <strong>DNA</strong> is required, add 20µl of RNase A (DNase-Free, 20mg/ml). Mix and incubate at 37°C for 10 min. 2. Homogenization Centrifuge at 12,000 x g for 5 min to pellet the cell debris and contaminants. Transfer 450µl of cleared supernatant to a clean microcentrifuge tube. Add 1.5 volumes of Buffer FB (~680µl without RNase A treatment, ~710µl with RNase A treatment) and mix thoroughly by pulsed vortexing until a homogenous solution is obtained. Incubate at 65°C for 10 min. When transferring the supernatant, avoid touching the pellet and the layer of contaminants on top of the solutions if any. If the supernatant transferred is less than 450µl, adjust the volume of Buffer FB to be added proportionately. 3. Addition of ethanol Add 350µl of absolute ethanol. Mix immediately and thoroughly by pulsed-vortexing to obtain homogenous solution. Centrifuge at 5,000 x g for 1 min. Mix immediately to prevent any uneven precipitation of nucleic acid due to high local ethanol concentrations. 4. Loading to column Attention! Tips for Higher Yield In order to obtain maximum yield, we strongly recommend users to fix the orientation of the column during centrifugation at all times. We recommend users to place the column which has a triangle mark on the edge, at a fixed position during centrifugation Transfer approximately 750µl of supernatant into a column assembled in a clean collection tube (provided). Centrifuge at 5,000 x g for 1 min. Discard flow through. Repeat for the remaining sample from step 3.
Page 2 and 3: Introduction The GF-1 Food DNA Extr
Page 6 and 7: 5. Column washing 1 Wash the column
Page 8: Problem Possibility Suggestions Col

Food DNA isolation miniprep kit: Manual

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