---++ Large Scale Metagenomic Soil Prep * Combine 80g soil with 87mL 0.1M pH 6.6 monobasic sodium phosphate in a beaker on a stir plate with a stir bar. Allow this to mix thoroughly via stirring for about 5 minutes. * Add 13mL 0.5M aluminum sulfate to the beaker. Watch out for excess foaming as gas is released from the soil-buffer mixture. Remove any non-dissolving white foam from the top of the mixture after the foam as broken, allowing the solution to continue to mix for at least 5 more minutes. * Using 6M sodium hydroxide, raise the pH of the soil mixture to pH 8.0. * Combine the pH 8.0 soil mixture with 100mL pH 8 lysis buffer (100mM NaCl, 500mM Tris (pH 8), 10% SDS w/v ) in the plastic Bead Beater mixing chamber. Add 0.1mm beads to the chamber until it no longer contains air (about 150mL of beads) when the beater propeller is positioned into the chamber. * Assemble the Bead Beater with the full mixing chamber. Make sure to fill the ice jacket with fresh ice and water. * Run the Bead Beater for 2 minutes. * Disassemble the Bead Beater and aliquot the lysed soil into 6-50mL conical flasks. * Spin the flasks down at 5000rpm in a fixed rotor centrifuge for 10 minutes. Pipet off the supernatant into new 50mL conical tubes (approx 2). * Pass the lysate through a #5 whatman filter, divide it into 10mL aliquots in new 50mL conicals. _For each aliquot:_ * Add 10mL chloroform:isoamyl alcohol 24:1. * Vortex well and let sit 4C for 15 minutes. * Spin down at 10,000rpm for 10 minutes. * Remove and reserve the aqueous phase (top layer) using a 10mL pipet, being very careful not to take up any of the organic phase or interphase (the layer of chunks between the two layers). * Repeat the centrifugation with the removed aqueous phase to ensure no residual chloroform had been left. * If no additional chloroform is found, leave the conical tube open in the fume hood until there is no residual smell of chloroform. If additional chloroform collects at the bottom of the tube, decant and repeat the centrifugation. Properly dispose of all chloroform waste. * Once all chloroform smell has dissipated, add 5mL 7.5M ammonium acetate and 10mL 100% isopropanol to the tubes. Vortex well. * Cover conicals and move them to -20C for at least 3 hours, preferably over night. * Spin down the precipitations at 10,000rpm for 30 minutes. * Decant the isopropanol/salt mixture, being careful not to disturb the DNA pellet. * Perform 3 washed with 5mL molecular grade 70% ethanol and a 10 minute 10,000rpm spin down each time. * After removing the final round of ethanol, allow the pellet to air dry in the fume hood until no ethanol remains in the tube. * Rehydrate the pellet with 500uL sterile dH2O. Vortex well to full resuspend the DNA * Pass the DNA solution through a Spin-X column via centrifugation to remove any remaining solid particulate or undissolved DNA. * Pool all DNA extractions. * Quantify dsDNA, store at -20C. -- Main.BrianRenda - 20 Jan 2012
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Topic revision: r1 - 2012-01-20 - BrianRenda