other:
Ligation:
Transfection:
Environmental Sampling:Article Title: Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules.
Article Snippet: .. .Environmental sample (1 kg of soil) .Lysis buffer: 100 mM Tris-HCl, 100 mM Na EDTA, 1.5 M NaCl, 1% (w/v) cetyl trimethyl ammonium bromide, 2% (w/v) SDS, pH 8.0 (this will form a slurry) .Isopropanol .70% ethanol .Lambda HindIII molecular weight standard .TE: 10 mM Tris, 1 mM EDTA, pH 8 .End-It DNA end-repair kit (Epicentre Biotechnologies) .3 M sodium acetate pH 5.0 .DNA ligase .Blunt-end-digested and dephosphorylated cosmid or fosmid (commercially prepared cosmids, pWEB and pWEB::TNC, are available from Epicentre Biotechnologies) .Lambda phage packaging extracts (Epicentre Biotechnologies or Stratagene) .Sterile phage dilution buffer: 10 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 8.3 .E. coli (most standard cloning strains of E. coli can be used, e.g., XL-1 Blue) .LB broth .1.0 M MgSO4 .0.5 TBE: 45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8 .150 mm LB agar plates with selection antibiotic (1 plate for every 1,500 clones to be screened) .Assay strain (B. subtilis transformed with a plasmid that confers resistance to the antibiotic used for selecting the cosmid library) .LB top agar .100 mm LB agar plates with selection and counter-selection antibiotics (LB plates with ampicillin will work if you are using pWEB or pWEB::TNC) .. .Coarse mesh screen (1/4 inch mesh can be purchased at a hardware store) .Fine mesh screen (1/8 inch mesh can be purchased at a hardware store) .500-ml wide-mouth high-density polyethylene bottles (Nalgene bottles) .500-ml centrifuge bottles .500 ml graduated cylinder .15 and 50 ml polypropylene centrifuge tubes (Falcon tubes) .Razor blade .Straight edge .Dish for staining edge gel slices with ethidium bromide .Dialysis tubing and clamps (10,000 MWCO, 32 mm) .Transfer pipettes .30,000 MWCO centrifugal concentrator (Amicon, 15 ml size) .Sterile repeating pump for measuring 15 ml .Sterile toothpicks .Preparative agarose gel apparatus (20 10 cm and free of ethidium bromide) .Comb with one large well (Z750 mm 10 mm 1 mm per well) .Mini agarose gel apparatus .UV light .Refrigerated microcentrifuge .37 1C shaker .37 and 30 1C incubators .30, 50 and 70 1C water baths .Centrifuge for 500 ml or 1 liter bottles .Centrifuge for 50 ml conical tubes .Dissecting microscope PROCEDURE eDNA isolation TIMING 5–6 h 1| Collect approximately 1 kg of organic-rich soil or mud.
Lysis:Article Title: Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules.
Article Snippet: .. .Environmental sample (1 kg of soil) .Lysis buffer: 100 mM Tris-HCl, 100 mM Na EDTA, 1.5 M NaCl, 1% (w/v) cetyl trimethyl ammonium bromide, 2% (w/v) SDS, pH 8.0 (this will form a slurry) .Isopropanol .70% ethanol .Lambda HindIII molecular weight standard .TE: 10 mM Tris, 1 mM EDTA, pH 8 .End-It DNA end-repair kit (Epicentre Biotechnologies) .3 M sodium acetate pH 5.0 .DNA ligase .Blunt-end-digested and dephosphorylated cosmid or fosmid (commercially prepared cosmids, pWEB and pWEB::TNC, are available from Epicentre Biotechnologies) .Lambda phage packaging extracts (Epicentre Biotechnologies or Stratagene) .Sterile phage dilution buffer: 10 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 8.3 .E. coli (most standard cloning strains of E. coli can be used, e.g., XL-1 Blue) .LB broth .1.0 M MgSO4 .0.5 TBE: 45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8 .150 mm LB agar plates with selection antibiotic (1 plate for every 1,500 clones to be screened) .Assay strain (B. subtilis transformed with a plasmid that confers resistance to the antibiotic used for selecting the cosmid library) .LB top agar .100 mm LB agar plates with selection and counter-selection antibiotics (LB plates with ampicillin will work if you are using pWEB or pWEB::TNC) .. .Coarse mesh screen (1/4 inch mesh can be purchased at a hardware store) .Fine mesh screen (1/8 inch mesh can be purchased at a hardware store) .500-ml wide-mouth high-density polyethylene bottles (Nalgene bottles) .500-ml centrifuge bottles .500 ml graduated cylinder .15 and 50 ml polypropylene centrifuge tubes (Falcon tubes) .Razor blade .Straight edge .Dish for staining edge gel slices with ethidium bromide .Dialysis tubing and clamps (10,000 MWCO, 32 mm) .Transfer pipettes .30,000 MWCO centrifugal concentrator (Amicon, 15 ml size) .Sterile repeating pump for measuring 15 ml .Sterile toothpicks .Preparative agarose gel apparatus (20 10 cm and free of ethidium bromide) .Comb with one large well (Z750 mm 10 mm 1 mm per well) .Mini agarose gel apparatus .UV light .Refrigerated microcentrifuge .37 1C shaker .37 and 30 1C incubators .30, 50 and 70 1C water baths .Centrifuge for 500 ml or 1 liter bottles .Centrifuge for 50 ml conical tubes .Dissecting microscope PROCEDURE eDNA isolation TIMING 5–6 h 1| Collect approximately 1 kg of organic-rich soil or mud.
Molecular Weight:Article Title: Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules.
Article Snippet: .. .Environmental sample (1 kg of soil) .Lysis buffer: 100 mM Tris-HCl, 100 mM Na EDTA, 1.5 M NaCl, 1% (w/v) cetyl trimethyl ammonium bromide, 2% (w/v) SDS, pH 8.0 (this will form a slurry) .Isopropanol .70% ethanol .Lambda HindIII molecular weight standard .TE: 10 mM Tris, 1 mM EDTA, pH 8 .End-It DNA end-repair kit (Epicentre Biotechnologies) .3 M sodium acetate pH 5.0 .DNA ligase .Blunt-end-digested and dephosphorylated cosmid or fosmid (commercially prepared cosmids, pWEB and pWEB::TNC, are available from Epicentre Biotechnologies) .Lambda phage packaging extracts (Epicentre Biotechnologies or Stratagene) .Sterile phage dilution buffer: 10 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 8.3 .E. coli (most standard cloning strains of E. coli can be used, e.g., XL-1 Blue) .LB broth .1.0 M MgSO4 .0.5 TBE: 45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8 .150 mm LB agar plates with selection antibiotic (1 plate for every 1,500 clones to be screened) .Assay strain (B. subtilis transformed with a plasmid that confers resistance to the antibiotic used for selecting the cosmid library) .LB top agar .100 mm LB agar plates with selection and counter-selection antibiotics (LB plates with ampicillin will work if you are using pWEB or pWEB::TNC) .. .Coarse mesh screen (1/4 inch mesh can be purchased at a hardware store) .Fine mesh screen (1/8 inch mesh can be purchased at a hardware store) .500-ml wide-mouth high-density polyethylene bottles (Nalgene bottles) .500-ml centrifuge bottles .500 ml graduated cylinder .15 and 50 ml polypropylene centrifuge tubes (Falcon tubes) .Razor blade .Straight edge .Dish for staining edge gel slices with ethidium bromide .Dialysis tubing and clamps (10,000 MWCO, 32 mm) .Transfer pipettes .30,000 MWCO centrifugal concentrator (Amicon, 15 ml size) .Sterile repeating pump for measuring 15 ml .Sterile toothpicks .Preparative agarose gel apparatus (20 10 cm and free of ethidium bromide) .Comb with one large well (Z750 mm 10 mm 1 mm per well) .Mini agarose gel apparatus .UV light .Refrigerated microcentrifuge .37 1C shaker .37 and 30 1C incubators .30, 50 and 70 1C water baths .Centrifuge for 500 ml or 1 liter bottles .Centrifuge for 50 ml conical tubes .Dissecting microscope PROCEDURE eDNA isolation TIMING 5–6 h 1| Collect approximately 1 kg of organic-rich soil or mud.
Sterility:Article Title: Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules.
Article Snippet: .. .Environmental sample (1 kg of soil) .Lysis buffer: 100 mM Tris-HCl, 100 mM Na EDTA, 1.5 M NaCl, 1% (w/v) cetyl trimethyl ammonium bromide, 2% (w/v) SDS, pH 8.0 (this will form a slurry) .Isopropanol .70% ethanol .Lambda HindIII molecular weight standard .TE: 10 mM Tris, 1 mM EDTA, pH 8 .End-It DNA end-repair kit (Epicentre Biotechnologies) .3 M sodium acetate pH 5.0 .DNA ligase .Blunt-end-digested and dephosphorylated cosmid or fosmid (commercially prepared cosmids, pWEB and pWEB::TNC, are available from Epicentre Biotechnologies) .Lambda phage packaging extracts (Epicentre Biotechnologies or Stratagene) .Sterile phage dilution buffer: 10 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 8.3 .E. coli (most standard cloning strains of E. coli can be used, e.g., XL-1 Blue) .LB broth .1.0 M MgSO4 .0.5 TBE: 45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8 .150 mm LB agar plates with selection antibiotic (1 plate for every 1,500 clones to be screened) .Assay strain (B. subtilis transformed with a plasmid that confers resistance to the antibiotic used for selecting the cosmid library) .LB top agar .100 mm LB agar plates with selection and counter-selection antibiotics (LB plates with ampicillin will work if you are using pWEB or pWEB::TNC) .. .Coarse mesh screen (1/4 inch mesh can be purchased at a hardware store) .Fine mesh screen (1/8 inch mesh can be purchased at a hardware store) .500-ml wide-mouth high-density polyethylene bottles (Nalgene bottles) .500-ml centrifuge bottles .500 ml graduated cylinder .15 and 50 ml polypropylene centrifuge tubes (Falcon tubes) .Razor blade .Straight edge .Dish for staining edge gel slices with ethidium bromide .Dialysis tubing and clamps (10,000 MWCO, 32 mm) .Transfer pipettes .30,000 MWCO centrifugal concentrator (Amicon, 15 ml size) .Sterile repeating pump for measuring 15 ml .Sterile toothpicks .Preparative agarose gel apparatus (20 10 cm and free of ethidium bromide) .Comb with one large well (Z750 mm 10 mm 1 mm per well) .Mini agarose gel apparatus .UV light .Refrigerated microcentrifuge .37 1C shaker .37 and 30 1C incubators .30, 50 and 70 1C water baths .Centrifuge for 500 ml or 1 liter bottles .Centrifuge for 50 ml conical tubes .Dissecting microscope PROCEDURE eDNA isolation TIMING 5–6 h 1| Collect approximately 1 kg of organic-rich soil or mud.
Cloning:Article Title: Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules.
Article Snippet: .. .Environmental sample (1 kg of soil) .Lysis buffer: 100 mM Tris-HCl, 100 mM Na EDTA, 1.5 M NaCl, 1% (w/v) cetyl trimethyl ammonium bromide, 2% (w/v) SDS, pH 8.0 (this will form a slurry) .Isopropanol .70% ethanol .Lambda HindIII molecular weight standard .TE: 10 mM Tris, 1 mM EDTA, pH 8 .End-It DNA end-repair kit (Epicentre Biotechnologies) .3 M sodium acetate pH 5.0 .DNA ligase .Blunt-end-digested and dephosphorylated cosmid or fosmid (commercially prepared cosmids, pWEB and pWEB::TNC, are available from Epicentre Biotechnologies) .Lambda phage packaging extracts (Epicentre Biotechnologies or Stratagene) .Sterile phage dilution buffer: 10 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 8.3 .E. coli (most standard cloning strains of E. coli can be used, e.g., XL-1 Blue) .LB broth .1.0 M MgSO4 .0.5 TBE: 45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8 .150 mm LB agar plates with selection antibiotic (1 plate for every 1,500 clones to be screened) .Assay strain (B. subtilis transformed with a plasmid that confers resistance to the antibiotic used for selecting the cosmid library) .LB top agar .100 mm LB agar plates with selection and counter-selection antibiotics (LB plates with ampicillin will work if you are using pWEB or pWEB::TNC) .. .Coarse mesh screen (1/4 inch mesh can be purchased at a hardware store) .Fine mesh screen (1/8 inch mesh can be purchased at a hardware store) .500-ml wide-mouth high-density polyethylene bottles (Nalgene bottles) .500-ml centrifuge bottles .500 ml graduated cylinder .15 and 50 ml polypropylene centrifuge tubes (Falcon tubes) .Razor blade .Straight edge .Dish for staining edge gel slices with ethidium bromide .Dialysis tubing and clamps (10,000 MWCO, 32 mm) .Transfer pipettes .30,000 MWCO centrifugal concentrator (Amicon, 15 ml size) .Sterile repeating pump for measuring 15 ml .Sterile toothpicks .Preparative agarose gel apparatus (20 10 cm and free of ethidium bromide) .Comb with one large well (Z750 mm 10 mm 1 mm per well) .Mini agarose gel apparatus .UV light .Refrigerated microcentrifuge .37 1C shaker .37 and 30 1C incubators .30, 50 and 70 1C water baths .Centrifuge for 500 ml or 1 liter bottles .Centrifuge for 50 ml conical tubes .Dissecting microscope PROCEDURE eDNA isolation TIMING 5–6 h 1| Collect approximately 1 kg of organic-rich soil or mud.
Selection:Article Title: Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules.
Article Snippet: .. .Environmental sample (1 kg of soil) .Lysis buffer: 100 mM Tris-HCl, 100 mM Na EDTA, 1.5 M NaCl, 1% (w/v) cetyl trimethyl ammonium bromide, 2% (w/v) SDS, pH 8.0 (this will form a slurry) .Isopropanol .70% ethanol .Lambda HindIII molecular weight standard .TE: 10 mM Tris, 1 mM EDTA, pH 8 .End-It DNA end-repair kit (Epicentre Biotechnologies) .3 M sodium acetate pH 5.0 .DNA ligase .Blunt-end-digested and dephosphorylated cosmid or fosmid (commercially prepared cosmids, pWEB and pWEB::TNC, are available from Epicentre Biotechnologies) .Lambda phage packaging extracts (Epicentre Biotechnologies or Stratagene) .Sterile phage dilution buffer: 10 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 8.3 .E. coli (most standard cloning strains of E. coli can be used, e.g., XL-1 Blue) .LB broth .1.0 M MgSO4 .0.5 TBE: 45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8 .150 mm LB agar plates with selection antibiotic (1 plate for every 1,500 clones to be screened) .Assay strain (B. subtilis transformed with a plasmid that confers resistance to the antibiotic used for selecting the cosmid library) .LB top agar .100 mm LB agar plates with selection and counter-selection antibiotics (LB plates with ampicillin will work if you are using pWEB or pWEB::TNC) .. .Coarse mesh screen (1/4 inch mesh can be purchased at a hardware store) .Fine mesh screen (1/8 inch mesh can be purchased at a hardware store) .500-ml wide-mouth high-density polyethylene bottles (Nalgene bottles) .500-ml centrifuge bottles .500 ml graduated cylinder .15 and 50 ml polypropylene centrifuge tubes (Falcon tubes) .Razor blade .Straight edge .Dish for staining edge gel slices with ethidium bromide .Dialysis tubing and clamps (10,000 MWCO, 32 mm) .Transfer pipettes .30,000 MWCO centrifugal concentrator (Amicon, 15 ml size) .Sterile repeating pump for measuring 15 ml .Sterile toothpicks .Preparative agarose gel apparatus (20 10 cm and free of ethidium bromide) .Comb with one large well (Z750 mm 10 mm 1 mm per well) .Mini agarose gel apparatus .UV light .Refrigerated microcentrifuge .37 1C shaker .37 and 30 1C incubators .30, 50 and 70 1C water baths .Centrifuge for 500 ml or 1 liter bottles .Centrifuge for 50 ml conical tubes .Dissecting microscope PROCEDURE eDNA isolation TIMING 5–6 h 1| Collect approximately 1 kg of organic-rich soil or mud.
Clone Assay:Article Title: Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules.
Article Snippet: .. .Environmental sample (1 kg of soil) .Lysis buffer: 100 mM Tris-HCl, 100 mM Na EDTA, 1.5 M NaCl, 1% (w/v) cetyl trimethyl ammonium bromide, 2% (w/v) SDS, pH 8.0 (this will form a slurry) .Isopropanol .70% ethanol .Lambda HindIII molecular weight standard .TE: 10 mM Tris, 1 mM EDTA, pH 8 .End-It DNA end-repair kit (Epicentre Biotechnologies) .3 M sodium acetate pH 5.0 .DNA ligase .Blunt-end-digested and dephosphorylated cosmid or fosmid (commercially prepared cosmids, pWEB and pWEB::TNC, are available from Epicentre Biotechnologies) .Lambda phage packaging extracts (Epicentre Biotechnologies or Stratagene) .Sterile phage dilution buffer: 10 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 8.3 .E. coli (most standard cloning strains of E. coli can be used, e.g., XL-1 Blue) .LB broth .1.0 M MgSO4 .0.5 TBE: 45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8 .150 mm LB agar plates with selection antibiotic (1 plate for every 1,500 clones to be screened) .Assay strain (B. subtilis transformed with a plasmid that confers resistance to the antibiotic used for selecting the cosmid library) .LB top agar .100 mm LB agar plates with selection and counter-selection antibiotics (LB plates with ampicillin will work if you are using pWEB or pWEB::TNC) .. .Coarse mesh screen (1/4 inch mesh can be purchased at a hardware store) .Fine mesh screen (1/8 inch mesh can be purchased at a hardware store) .500-ml wide-mouth high-density polyethylene bottles (Nalgene bottles) .500-ml centrifuge bottles .500 ml graduated cylinder .15 and 50 ml polypropylene centrifuge tubes (Falcon tubes) .Razor blade .Straight edge .Dish for staining edge gel slices with ethidium bromide .Dialysis tubing and clamps (10,000 MWCO, 32 mm) .Transfer pipettes .30,000 MWCO centrifugal concentrator (Amicon, 15 ml size) .Sterile repeating pump for measuring 15 ml .Sterile toothpicks .Preparative agarose gel apparatus (20 10 cm and free of ethidium bromide) .Comb with one large well (Z750 mm 10 mm 1 mm per well) .Mini agarose gel apparatus .UV light .Refrigerated microcentrifuge .37 1C shaker .37 and 30 1C incubators .30, 50 and 70 1C water baths .Centrifuge for 500 ml or 1 liter bottles .Centrifuge for 50 ml conical tubes .Dissecting microscope PROCEDURE eDNA isolation TIMING 5–6 h 1| Collect approximately 1 kg of organic-rich soil or mud.
Transformation Assay:Article Title: Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules.
Article Snippet: .. .Environmental sample (1 kg of soil) .Lysis buffer: 100 mM Tris-HCl, 100 mM Na EDTA, 1.5 M NaCl, 1% (w/v) cetyl trimethyl ammonium bromide, 2% (w/v) SDS, pH 8.0 (this will form a slurry) .Isopropanol .70% ethanol .Lambda HindIII molecular weight standard .TE: 10 mM Tris, 1 mM EDTA, pH 8 .End-It DNA end-repair kit (Epicentre Biotechnologies) .3 M sodium acetate pH 5.0 .DNA ligase .Blunt-end-digested and dephosphorylated cosmid or fosmid (commercially prepared cosmids, pWEB and pWEB::TNC, are available from Epicentre Biotechnologies) .Lambda phage packaging extracts (Epicentre Biotechnologies or Stratagene) .Sterile phage dilution buffer: 10 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 8.3 .E. coli (most standard cloning strains of E. coli can be used, e.g., XL-1 Blue) .LB broth .1.0 M MgSO4 .0.5 TBE: 45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8 .150 mm LB agar plates with selection antibiotic (1 plate for every 1,500 clones to be screened) .Assay strain (B. subtilis transformed with a plasmid that confers resistance to the antibiotic used for selecting the cosmid library) .LB top agar .100 mm LB agar plates with selection and counter-selection antibiotics (LB plates with ampicillin will work if you are using pWEB or pWEB::TNC) .. .Coarse mesh screen (1/4 inch mesh can be purchased at a hardware store) .Fine mesh screen (1/8 inch mesh can be purchased at a hardware store) .500-ml wide-mouth high-density polyethylene bottles (Nalgene bottles) .500-ml centrifuge bottles .500 ml graduated cylinder .15 and 50 ml polypropylene centrifuge tubes (Falcon tubes) .Razor blade .Straight edge .Dish for staining edge gel slices with ethidium bromide .Dialysis tubing and clamps (10,000 MWCO, 32 mm) .Transfer pipettes .30,000 MWCO centrifugal concentrator (Amicon, 15 ml size) .Sterile repeating pump for measuring 15 ml .Sterile toothpicks .Preparative agarose gel apparatus (20 10 cm and free of ethidium bromide) .Comb with one large well (Z750 mm 10 mm 1 mm per well) .Mini agarose gel apparatus .UV light .Refrigerated microcentrifuge .37 1C shaker .37 and 30 1C incubators .30, 50 and 70 1C water baths .Centrifuge for 500 ml or 1 liter bottles .Centrifuge for 50 ml conical tubes .Dissecting microscope PROCEDURE eDNA isolation TIMING 5–6 h 1| Collect approximately 1 kg of organic-rich soil or mud.
Plasmid Preparation:Article Title: Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules.
Article Snippet: .. .Environmental sample (1 kg of soil) .Lysis buffer: 100 mM Tris-HCl, 100 mM Na EDTA, 1.5 M NaCl, 1% (w/v) cetyl trimethyl ammonium bromide, 2% (w/v) SDS, pH 8.0 (this will form a slurry) .Isopropanol .70% ethanol .Lambda HindIII molecular weight standard .TE: 10 mM Tris, 1 mM EDTA, pH 8 .End-It DNA end-repair kit (Epicentre Biotechnologies) .3 M sodium acetate pH 5.0 .DNA ligase .Blunt-end-digested and dephosphorylated cosmid or fosmid (commercially prepared cosmids, pWEB and pWEB::TNC, are available from Epicentre Biotechnologies) .Lambda phage packaging extracts (Epicentre Biotechnologies or Stratagene) .Sterile phage dilution buffer: 10 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 8.3 .E. coli (most standard cloning strains of E. coli can be used, e.g., XL-1 Blue) .LB broth .1.0 M MgSO4 .0.5 TBE: 45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8 .150 mm LB agar plates with selection antibiotic (1 plate for every 1,500 clones to be screened) .Assay strain (B. subtilis transformed with a plasmid that confers resistance to the antibiotic used for selecting the cosmid library) .LB top agar .100 mm LB agar plates with selection and counter-selection antibiotics (LB plates with ampicillin will work if you are using pWEB or pWEB::TNC) .. .Coarse mesh screen (1/4 inch mesh can be purchased at a hardware store) .Fine mesh screen (1/8 inch mesh can be purchased at a hardware store) .500-ml wide-mouth high-density polyethylene bottles (Nalgene bottles) .500-ml centrifuge bottles .500 ml graduated cylinder .15 and 50 ml polypropylene centrifuge tubes (Falcon tubes) .Razor blade .Straight edge .Dish for staining edge gel slices with ethidium bromide .Dialysis tubing and clamps (10,000 MWCO, 32 mm) .Transfer pipettes .30,000 MWCO centrifugal concentrator (Amicon, 15 ml size) .Sterile repeating pump for measuring 15 ml .Sterile toothpicks .Preparative agarose gel apparatus (20 10 cm and free of ethidium bromide) .Comb with one large well (Z750 mm 10 mm 1 mm per well) .Mini agarose gel apparatus .UV light .Refrigerated microcentrifuge .37 1C shaker .37 and 30 1C incubators .30, 50 and 70 1C water baths .Centrifuge for 500 ml or 1 liter bottles .Centrifuge for 50 ml conical tubes .Dissecting microscope PROCEDURE eDNA isolation TIMING 5–6 h 1| Collect approximately 1 kg of organic-rich soil or mud.
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