Technique / Molecular Biology / DNA analysis techniques / DNA extraction purification and quantification

Genomic DNA extraction protocol

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By egale on 20-Sep-2006 DNA Isolation from Fixed or Paraffin-embedded Tissue Protocol * Lysis buffer 10mM Tris-Cl (pH8.0) 0.1M EDTA(pH8.0) 0.5%(w/v)SDS 20 ug/ml DNase-free pancreatic RNase * Xylenes (Fisher cat. no. X5-500) * Isopropanol (2-propanol)(Fisher cat. no. A464-4) * 70% Ethanol * 1.5 ml Sterile microfuge tubes, or 15ml centrifuge tubes * Micropipettors and pipets * Vortex mixer * Microfuge or clinical centrifuge * Water bath DNA Isolation from Paraffin-embedded Tissue Paraffin-embedded Tissue—Sample De-paraffinization 1.Place 50-micron thick scrolls (weight up to 5-10 mg), finely minced, into a 1.5 ml microfuge tube. Add 300 ul Xylene and incubate 5 minutes with constant gentle mixing (for example on a lab rotator) at room temperature. 2.Centrifuge at 13,000-16,000 x g for 1-3 minutes to pellet the tissue. Discard the Xylene. 3.Repeat steps 1- 2, twice (for a total of three Xylene washes). 4.Add 300 ul 100% Ethanol and incubate 5 minutes with constant mixing at room temperature. 5.Centrifuge at 13,000-16,000 x g for 1-3 minutes to pellet the tissue. Discard the ethanol. 6. Repeat steps 4-5 (for a total of two ethanol washes). Cell Lysis: 1.Add 10 volume deparaffined tissues(V/W) of lysis buffer(aroud 100-300 ul) in sample tube and proteinase K(20mg/ml) to a final lysis buffer, incubate 3 hours at 50C. Swirl tube from time and time. 2.Treatment with phenol Add equal volume phenol, gently mix 10 minutes and centrifuge 10 minutes at 5000g at RT.Seperate two phases. 3.Use a pipette to transfer the supernanant.(aqueous phases) repeat extraction with phenol. 4.Transfer aqueous phase to a new tube, add 0.2 volume of 10 M ammonium acetate, and add 2.5 volume 100% ethanol at RT. Mix well.See DNA fragments, centrifuge at 5000g. if not see DNA, freeze tube at -20C over night. centifuge later on. 5. wash with 70% ethanol twice 6. Disslove DNA with 50 ul TE buffer. 7.Store DNA at 4 °C. For long term storage, place sample at -20°C or -80°C. Rating: Very Good Reply