Preparation of genomic DNA from frozen tissue

  • 1st day:
    • 200...500 ng (about 1/4 cm3) tissue sample stored at - 80°C
    • add 1 ml lysis buffer (20 mM TRIS pH 8; 1mM EDTA; 100mM NaCl; 0.5% SDS)
    • add 20µl (= 0.3 mg) Protease K
    • shake ON at 55°C
     
  • 2nd day:
    • 2 x extraction with equal volume PhOH/CHCl3
    • 2 x extraction with equal volume CHCl3
    • ppt. with equal volume Isopropanol/NaOAc at RT
    • 30 min centrifugation
    • wash with 1 ml 70% EtOH
    • air dry
    • resolve in 200 µl 1 mM TRIS pH 8 and shake for 2 hours at 4°C to resolve the DNA
    • 2 µl on 1% TAE agarose gel for quality control and quantification