Soil-based dna extraction

Abstract

Methods of extracting DNA from soil involve lysing microbial cells contained within the soil by mixing it with an extraction buffer containing cetrimonium bromide (cTAB). The cTAB helps reduce or eliminate high levels of humic acid often present within the soil, which interferes with processes including PCR. Methods further involve binding DNA lysed from the microbial cells and then bound to a silica substrate, washing non-DNA debris from the silica substrate, eluting the DNA from the substrate, and eluting the isolated DNA in an elution buffer. Example methods may involve extracting microbial DNA from a plurality of soil samples. Such methods involve adding soil samples into separate wells within a multi-well plate, lysing microbial cells within the samples using an extraction buffer, binding the microbial DNA released from the cells to silica particles, washing non-DNA debris from the silica particles, and separating the DNA from the silica particles.

What is claimed is: 
     
         1 . A method of extracting and isolating DNA (deoxyribonucleic acid) from a soil sample, the method comprising:
 using a first centrifuge plate or tube to:
 receive a soil suspension of the soil sample and a buffer; 
 lyse microbial cells within the soil suspension to form a lysis suspension; and 
 separate solid soil debris from DNA released from the lysed microbial cells, 
 wherein the DNA released from the lysed microbial cells is contained in a DNA supernatant; and 
   using a second centrifuge plate or tube to:
 receive a silica solution of suspended silica particles in water; 
 receive the DNA supernatant directly from the first centrifuge plate or tube; 
 admix the DNA supernatant with the silica solution, wherein the DNA released from the microbial cells binds to the suspended silica particles; 
 wash non-DNA debris from the silica particles; 
 isolate the DNA from the silica particles; and 
 elute the isolated DNA in an elution buffer in the second centrifuge plate or tube, 
 wherein the first centrifuge plate or tube and the second centrifuge plate or tube do not comprise disposable bead tubes, membranes, or spin columns. 
   
     
     
         2 . The method of  claim 1 , wherein the soil sample comprises about 250 μg to about 750 μg of soil. 
     
     
         3 . The method of  claim 1 , wherein a volume of the soil suspension is about 2 mL. 
     
     
         4 . The method of  claim 1 , wherein the silica solution further comprises about 6M guanidine thiocyanate. 
     
     
         5 . The method of  claim 1 , wherein the first centrifuge plate or tube is sonicated to lyse the microbial cells. 
     
     
         6 . The method of  claim 1 , wherein the buffer excludes proteinase K. 
     
     
         7 . The method of  claim 1 , wherein the buffer excludes at least one reagent selected from the group consisting of sodium dodecyl sulfate, one or more lysozymes, and glusulase. 
     
     
         8 . The method of  claim 1 , wherein the silica solution consists of suspended silica particles and water. 
     
     
         9 . The method of  claim 1 , wherein the silica solution excludes potassium iodide. 
     
     
         10 . The method of  claim 1 , wherein the first centrifuge plate or tube is used to lyse the microbial cells without any metal beads or grinding. 
     
     
         11 . A method of extracting and isolating DNA (deoxyribonucleic acid) from a plurality of soil samples, the method comprising:
 using a first centrifuge plate to:
 receive, in separate wells of the first centrifuge plate, a buffer and a soil suspension of each of the plurality of soil samples; 
 lyse microbial cells within the soil suspension to form a lysis suspension; and 
 separate solid soil debris from DNA released from the lysed microbial cells, 
 wherein the DNA released from the lysed microbial cells is contained in a DNA supernatant; and 
   using a second centrifuge plate to:
 receive a silica solution of suspended silica particles in water; 
 receive, in separate wells of the second centrifuge plate containing the silica solution, the DNA supernatant directly from the first centrifuge plate; 
 admix the DNA supernatant with the silica solution, wherein the DNA released from the microbial cells binds to the suspended silica particles; 
 wash non-DNA debris from the silica particles; 
 isolate the DNA from the silica particles; and 
 elute the isolated DNA in an elution buffer in the second centrifuge plate, 
 wherein the first centrifuge plate and the second centrifuge plate do not comprise disposable bead tubes, membranes, or spin columns. 
   
     
     
         12 . The method of  claim 11 , wherein each of the separate wells of the first centrifuge plate has a capacity of about 8 mL or about 10 mL. 
     
     
         13 . The method of  claim 11 , wherein each of the separate wells of the first centrifuge plate has a capacity of about 2 mL. 
     
     
         14 . The method of  claim 11 , wherein the first centrifuge plate and the second centrifuge plate are reusable. 
     
     
         15 . A kit for extracting and isolating DNA (deoxyribonucleic acid) from a soil sample, the kit comprising:
 a first reusable centrifuge plate or tube configured to receive a soil suspension of the soil sample;   a sonication buffer configured to lyse microbial cells within the soil suspension to form a lysis suspension in the first reusable centrifuge plate or tube;   a second reusable centrifuge plate or tube configured to receive DNA supernatant directly from the first reusable centrifuge plate or tube, the DNA supernatant derived from the lysis suspension;   a silica solution of suspended silica particles in water configured to bind DNA within the DNA supernatant in the second reusable centrifuge plate or tube;   a wash buffer configured to wash non-DNA debris bound to the silica particles in the second reusable centrifuge plate or tube; and   a DNA elution buffer configured to elute the isolated DNA in the second reusable centrifuge plate or tube,   wherein the kit is configured to isolate DNA from the soil sample without disposable bead tubes, membranes, or spin columns.   
     
     
         16 . The kit of  claim 15 , wherein the silica solution excludes potassium iodide. 
     
     
         17 . The kit of  claim 15 , wherein the first reusable centrifuge plate or tube is a reusable centrifuge plate comprising a plurality of wells each having a capacity of about 8 mL or about 10 mL. 
     
     
         18 . The kit of  claim 15 , wherein the sonication buffer comprises NaCl, polyvinylpyrrolidone, TE, RNaseA, and cTAB. 
     
     
         19 . The kit of  claim 15 , wherein the sonication buffer excludes proteinase K and sodium dodecyl sulfate. 
     
     
         20 . The kit of  claim 15 , wherein the kit excludes metal beads.