11.3. DNA extraction from various tissues for methylation analysis

Methylation analyses do not depend on a particular DNA extraction method. Nevertheless, the below extraction has been validated in a variety of honey bee tissues.

  1. Homogenize tissues in a 1.5 ml microcentrifuge tube in a small volume of NTE buffer:
    100 mM NaCl,
    50 mM Tris pH 8.2,
    10 mM EDTA,
    1% SDS,
    Proteinase K (500 µg per ml, freshly dissolved).
  2. Add a small amount (0.01%) of a non-ionic detergent such as Triton X100.
    The detergent is beneficial (increases the efficiency of proteinase K digestion), but not necessary.
  3. Add more buffer (roughly 500 µl per 20-50 mg of tissue).
  4. Incubate at 55°C for 1-3 hrs.
  5. Extract with 1 volume of phenol: chloroform.
  6. Spin for 10 min at 10,000rpm.
  7. Collect the upper phase (repeat the extraction if the upper phase looks cloudy).
  8. Add 1 ml of RNase A (10 mg/ml).
  9. Incubate for 10 min at 370C to digest RNA.
    This step is not necessary for bisulfite conversion, but the presence of RNA interferes with measuring DNA yield.
  10. Precipitate DNA with 1 volume of isopropanol or 2 volumes of EtOH.
  11. Spin gently (5,000 rpm for 2 min).
  12. Discard the supernatant.
  13. Wash the pellet once with 70% EtOH.
  14. Remove ethanol, but DO NOT DRY THE PELLET!
  15. Dissolve the pellet in TE buffer by heating to 65°C.
  16. Store at 4°C (or at -80°C for long term storage).

 

Clean DNA preps are stable at 4°C for at least 5 years. The majority of DNA strands from the above prep are 200-250kb in length with the smallest molecules around 70kb.

Note: DNA preps from larvae might appear milky after this procedure, but such preps are suitable for bisulfite conversions. Alternatively use the MasterPure DNA Purification kit from AMRESCO (Cat. No. MCD85201) yields cleaner larval preps.