Isolation of Cotton Genomic DNA from Leaf Tissue

Isolation of Cotton Genomic DNA from Leaf Tissue

(Kohel Lab Method as modified by Z. Yu, November, 1994)
  1. Use fresh [BEST] or Lyophilize the leaf tissue.
  2. Grind about 5 g leaf tissue with mortar and pestle in liquid N2, transfer the powder into an 50 ml polypropylene tube (50 ml) and store at -20°C.
  • Add 25 ml of ice-cold Extraction Buffer, mix well, and put on ice until centrifugation at 3750 rpm for 20 minutes (4°C); remove supernatant [Use swinging bucket rotor.].
  • Add 10 ml Lysis Buffer and vortex to resuspend pellet; incubate in at 65°C water bath for 30 minutes. Mix the tubes periodically by stirring or rocking the tubes every 10 minutes.
  • Add 12 ml of chloroform:octanol (24:1) and mix gently , inverting tube, until an emulsion forms.
  • Centrifuge at 3750 rpm for 20 minutes (15°C). Transfer upper phase to new 50 ml tube through Miracloth.
  • Add equal volume of cold isopropanol; mix gently; and let set at room temperature for more than 1 hour.
  • Centrifuge at 2500 rpm for 10 minutes (RT). Decant supernatant, and let tubes air dry. Then add 10 ml cold 76% etahnol/0.2 M Na Acetate. Put at 4°C for 1 hour or overnight. (good stopping point).
  • Centrifuge tubes (3750 rpm, 10 min). Carefully discard supernatent, and rinse the pellet with cold 70% EtOH. Let tubes dry upside down on paper towels (Keep track of tube labels!).
  • Let pellets dissolve into 3 ml TE buffer. Vortex at speed 5, and incubate at 65°C for about 1 hour.
  • Centrifuge at 3750 rpm for 10 minutes (15°C). Transfer supernatant to new 15 ml tube. Add RNase to a concentration of 20 µg/ml, mix gently and incubate at 37°C for 15 minutes.
  • Add 0.1 volume of 3M Na Acetate and two volumes of 95% ethanol for DNA precipitation. Place at -20°C for 1-12 hours (overnight).
  • Spool/scoop out the DNA on a 9" glass-hook pipet (new and unused) and let it air-dry under the hood or vacuum. If DNA cannot be spooled, centrifuge at 2500 rpm for 10 min to form DNA into pellet. Once pellet is dry, place pellet into 0.5 ml TE buffer in 1.5 ml microfuge tube for 30 min at 65°C.
  • Determine both concentration and quality of the DNA with spectrophotometer with a 40µl/800µl dilution (1/20), and by running digested and undigetsted DNA in a 1% agarose gel.

    Extraction Buffer (pH 6.0)

    • 0.35 M glucose
    • 0.1 M Tris HCl (pH 8.0)
    • 5.0 mM Na-EDTA (pH 8.0)
    • 2% PVP 40,000 MW
    • 0.1% DIECA, diethyldithiocarbamic acid (disodium salt)
    • 0.2% beta-mercapto-ethanol or Na2S2O5 (add when used)
    Lysis Buffer

    • 0.1 M Tris HCl (pH 8.0)
    • 1.4 M NaCl
    • 20 mM Na EDTA (pH 8.0)
    • 2% CTAB
    • 2% PVP 40,000 MW
    • 0.1% DIECA, diethyldithiocarbamic acid (disodium salt)
    • 0.2% beta-mercapto-ethanol or Na2S2O5 (add when used)