MET005 Soil DNA Extraction (Modified Method)
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1/4 Protocol Code: MET005 Version: 1.0 Date: 17-Apr-2013 Writer: Tran Nhat Anh / GEL Notes: For Metagenomic project Preparation Materials/Equipments 1 Soil sample 2 Medium 3 Antibiotic 4 100 mL flask 5 Petri dish Procedure S 1 Weighed 0.5 g of soil and put into a 2 mL centrifuge tube. 2 3 4 5 6 7 8 9 10 11 Dry the pellet in SpeedVac for 15 min. 12 Dissolve the DNA in 20 µL UltraPure water. K 1 2 Purify the solution with Qiagen kit. Soil DNA extraction and purification (M method) Add 0.6 mL extraction buffer, 0.3 mL of phenol-chloroform- alcohol (25:24:1) and 0.5 g glass beads into the tube. Vor Centrifuge the homogenate at 16000 g for 2 min. Coll Mix supernatant with the same volume of phenol-chloro isoamyl alcohol (25:24:1) and centrifuged at 6000 g f Mix supernatant with the equal volume of chloroform-i alcohol (24:1) and centrifuge at 16000 g for 5 min. C To the supernatant, add 0.3 mL extraction buffer con 4.5 M NaCl and 3% CTAB an incubate at 65°C for 30 min Mix the cooled incubated solution with an equal volum chloroform-isoamyl alcohol (24:1) and centrifuge at 3 Mix supernatant with 0.6 volume isopropanol. Incubate temperature for 30 min. Centrifuge the incubated solution at 16000 g for 15 m Collect the pellet. Mix the pellet with 1.5 mL of cold 70% ethanol and ce at 16000 g for 10 min. Collect the pellet. Add an equal volume of 1M CaCl2 in 1M HEPES-NaOH to t dissolved in water and incubate at room temperature f
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soil dna extraction
Transcript of MET005 Soil DNA Extraction (Modified Method)
1.1ProtocolSoil DNA extraction and purification (Modified method)Code:MET005Version:1.0Date:17-Apr-2013Writer:Tran Nhat Anh / GELNotes:For Metagenomic projectPreparationMaterials/EquipmentsQuantities (1 sample)1Soil sample0.125g2Medium40mL (each)3Antibiotic4100 mL flask4piece5Petri dish4pieceProcedureTotal timeS1Weighed 0.5 g of soil and put into a 2 mL centrifuge tube.2Add 0.6 mL extraction buffer, 0.3 mL of phenol-chloroform-isoamyl alcohol (25:24:1) and 0.5 g glass beads into the tube. Vortex at maximum speed for 2 min.3Centrifuge the homogenate at 16000 g for 2 min. Collect supernatant.4Mix supernatant with the same volume of phenol-chloroform-isoamyl alcohol (25:24:1) and centrifuged at 6000 g for 5 min. Collect supernatant.5Mix supernatant with the equal volume of chloroform-isoamyl alcohol (24:1) and centrifuge at 16000 g for 5 min. Collect supernatant.6To the supernatant, add 0.3 mL extraction buffer containing 4.5 M NaCl and 3% CTAB an incubate at 65C for 30 min.7Mix the cooled incubated solution with an equal volume of chloroform-isoamyl alcohol (24:1) and centrifuge at 3400 g for 20 min. Collect supernatant.8Mix supernatant with 0.6 volume isopropanol. Incubate at room temperature for 30 min.9Centrifuge the incubated solution at 16000 g for 15 min. Collect the pellet.10Mix the pellet with 1.5 mL of cold 70% ethanol and centrifuge at 16000 g for 10 min. Collect the pellet.11Dry the pellet in SpeedVac for 15 min.12Dissolve the DNA in 20 L UltraPure water.
K1Add an equal volume of 1M CaCl2 in 1M HEPES-NaOH to the DNA dissolved in water and incubate at room temperature for 30 min.2Purify the solution with Qiagen kit.
AppendixTable 1: Extraction bufferMaterialsFinal concQuantities (in 500 mL)1Tris-HCl500 mM39.5g2NaCl50 mM1.46g3Na2HPO43.3g4NaH2PO40.204g520% SDS5%125mL6DI water375mLAdjust pH to 8.0 using HCl/KOH
Table 2: Extraction buffer contain 4.5M NaCl and 3% CTABMaterialsFinal concQuantities (in 50mL)1CTAB5%1.5g2NaCl4.5 M13g2Extraction buffer50mLTable 5: 1M CaCl2MaterialsFinal concQuantities (in 10mL)1CaCl21M1.1g2HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid)1M2.38g31M NaOH in UltraPure water1.375mL4UltraPure water8.6mLAdjust pH to 7 with 1M NaOH and sterilize through a 0.22m filter
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