FastDNA™ SPIN Kit for Soil | Protocole détaillé

PROTOCOL Cat. No. 116560200/116560300 Revision Date: 2021-05 FastDNA™ SPIN Kit for Soil IMPORTANT? Before using SEWS-M wash solution, add 100 mL of 100% ethanol and mark on the bottle label the date ethanol was added. Add up to 500 mg of soil sample to a Lysing Matrix E tube. 1 NOTE? See section 3 in the User Manual for other important guidelines Add 978 ?L Sodium Phosphate Buffer to sample in Lysing 2 Matrix E tube. 3 Add 122 ?L MT Buffer to solubilize external contaminants. Homogenize in the FastPrep instrument for 40 seconds at a speed 4 setting of6.0 m/s to disrupt cell wall and release nucleic acids. Centrifuge at 14,000 x g for 5–10 minutes to pellet debris, such as insoluble cellular debris and lysing matrix. 5 NOTE? Extending centrifugation to 15 minutes can enhance elimination of excessive debris from large samples or from cells with complex cell walls. Transfer supernatant to a clean 2.0 mL microcentrifuge tube. Add 250 ?L PPS to separate the solubilized nucleic acids from 6 the cellular debris and lysing matrix. Mix by inverting the tube 10 times. Centrifuge at 14,000 x g for 5 minutes to pellet precipitate, removing the cellular debris and lysing matrix. Transfer supernatant to a clean 15 mL microcentrifuge tube. 7 NOTE? While a 2.0 mL microcentrifuge tube may be used at this step, more efficient mixing and DNA binding will occur in a larger tube. Resuspend the Binding Matrix suspension and add 1.0 mL 8 to the supernatant in the 15 mL tube.  PROTOCOL: FastDNA™ SPIN Kit for Soil Place on rotator or invert by hand for 2 minutes to allow binding 9 of DNA to the Binding Matrix. Place tube on a rack for 3 minutes to allow settling of Binding Matrix. Remove and discard 500 ?L of supernatant being careful to 10 avoid settled Binding Matrix. Gently resuspend Binding Matrix in the remaining amount of supernatant. Transfer approximately 600 ?L of the mixture to a 11 SPIN filter and centrifuge at14,000 x g for 1 minute. Empty the catch tube and reuse. Then repeat with the remaining mixture. Add 500 ?L prepared SEWS-M (with the appropriate amount of 12 ethanol added) to further solubilize impurities. Gently resuspend the pellet using the force of the liquid from the pipet tip. Centrifuge at 14,000 x g for 1 minute to remove impurities. 13 Empty the catch tube and reuse. Without addition of any liquid, centrifuge a second time at 14,000 x g for 2 minutes to “dry” the Binding Matrix of 14 residual wash solution. Replace the catch tube with a new, clean catch tube. 15 Air dry the SPIN Filter for 5 minutes at room temperature. Gently resuspend Binding Matrix (above the SPIN Filter) with 50–100 ?L of DES. 16 NOTE? To avoid over-dilution of the purified DNA, use smallest amount of DES required to resuspend Binding Matrix pellet. NOTE? Yields may be increased by incubation for 5 minutes at 55 °C in a heat block or water bath. Centrifuge at 14,000 x g for 1 minute to bring eluted DNA into 17 the clean catch tube. Discard the SPIN Filter. DNA is now ready for your analysis and downstream applications. Store at -20 °C for extended periods or 4 °C until use. MP BIOMEDICALS Learn more at www.mpbio.com