Extracting DNA from Plants for DArT

This is an alternative method developed by one of our collaborators, Karen James, Department of Botany, The Natural History Museum, London.

Solutions

  • CTAB buffer: 2% CTAB in 100Mm TRIS-HCL pH 8.0, 1.4M NaCL, 20mM EDTA
  • SARKOSYL buffer: 10% N-LAURYL SARCOSINE, 100mM TRIS-HCL pH8.0, 20mM EDTA
  • SEVAC: CHLOROFORM:ISOAMYL ALCOHOL 24:1
  • 0.1M T.E. BUFFER: 100Mm TRIS-HCL pH 8.0, 20mM EDTA

Steps

  1. Hand-grind plant material together with equal volume of acid-washed sand and liquid nitrogen using an acid-washed
    and autoclaved mortar & pestle (or 1.5-mL tube and micro-pestle) until the material reaches a chalky consistency.
    Transfer to 1.5-mL tube if necessary

  2. Add 500ul CTAB buffer and 50ul of SARKOSYL buffer, both pre-warmed to 60 degrees

  3. Add 10ul 10mg/mL PROTEINASE K

  4. Vortex vigorously & incubate for 60 minutes @ 60ºC with occasional vortexing

  5. SEVAC extraction:

    1. Add an equal volume of SEVAC and vortex vigorously
    2. Centrifuge top speed 3 minutes
    3. Transfer supernatant into a new tube without disturbing the interface
  6. Repeat the SEVAC extraction

  7. Add 2/3 volume (approx. 400ul) ice-cold isopropanol. Mix by gentle inversion and leave on ice for 30 min.

  8. Centrifuge 3 min and remove isopropanol to leave DNA pellet in bottom of tube

  9. Air dry pellet at 60 degrees or room temp (can stop here overnight or longer @ -20ºC)

  10. Re-suspend pellet in 32ul 0.1M T.E. buffer, run out 2uL on a 1% agarose gel

  11. Transfer 10ul into a different tube and store at –20 for safe keeping

  12. Bring vol of remaining 20uL DNA up to 200UL by adding 180uL 0.1X TE

  13. Phenol-chloroform purification (in fume hood):

    1. Add 100uL each of PHENOL and SEVAC and invert gently 1 minute
    2. Centrifuge top speed 10 minutes
    3. Transfer aqueous (top) layer into a new tube without disturbing the interface
    4. Add 200uL SEVAC and invert gently for 1 minute.
    5. Centrifuge at top speed for 10 minutes
    6. Transfer aqueous (top) layer into a new tube without disturbing the interface
  14. Add 20uL 3M SODIUM ACETATE pH 5.5 then 2X volumes (400ul) ice-cold 100% ETHANOL, mix gently by inversion and incubate at -20ºC overnight or one hour @ –80ºC

  15. Centrifuge @ 4ºC for 15 minutes then remove ethanol carefully with a pipette

  16. Wash with 500ul 70% ETHANOL by gentle inversion and centrifuge @ 4ºC for 5 minutes full speed

  17. Carefully remove ethanol with a pipette and air dry pellet in 60 degree heat block

  18. Re-suspend pellet in 30ul 0.1X TE (at 60C if necessary to dissolve difficult pellets)

  19. Run out 2uL on a 1% agarose gel

  20. Test DNA quality by restriction digest. Add the following, IN ORDER, to each restriction digest tube (keep reagents, especially the enzyme on ice at all times). Also set up a restriction digest of the concentration standard pGEM (20ng/uL), or some other comparable standard. Be sure to set up a no-enzyme control digest for each DNA sample to run out alongside your digest.

    RESTRICTION DIGEST

    • 5.8uL H20
    • 1uL 10X buffer (buffer “2” from NEB)
    • 2uL DNA1
    • 1uL 10X BSA
    • Mix well, then add: 0.2uL 50u/uL MseI (from NEB)

    NO-ENZYME CONTROL

    • 6uL H20
    • 1uL 10x buffer (buffer “2” from NEB)
    • 2uL DNA1
    • 1uL 10X BSA
    • Mix well.

    Mix gently by inverting and tapping tube on bench-top, then gently spin down contents of tubes and incubate for 2 hours at 37ºC. Add 2uL loading dye to each tube and run whole reaction out on 1% Agarose gel for at least 1 hour at 40 Volts.

  21. Estimate DNA concentration by comparing un-digested DNA to un-digested pGem on the gel. DArT requires at least 1ug DNA at approx 100ng/uL concentration

1How much DNA to use is a matter of judgement. 2uL is about right if you can see your high molecular weight DNA as a discrete band on your gel. If you have a very bright smear of DNA which masks your high molecular weight band, try reducing your DNA to 1uL; if you can barely see your high molecular weight band, increase your DNA volume. For any adjustment in DNA volume, adjust water accordingly so that reaction volume is always 10uL

Page last updated: March 26, 2019, 1:02 pm