Jimenez-gomez lab:protocol CTAB 96 well DNA extraction

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Institute Jean Pierre Bourgin
INRA - Versailles
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78026 Versailles Cedex
France
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Max Planck Institute for Plant Breeding Research
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96-Well Format DNA Extraction with CTAB


 

  1. Add a single ball bearing to each 1.2 ml tube containing tissue. (This can be done by using a sealing mat with 96 indentations and sprinkling the ball bearings over the top). Put samples into liquid nitrogen.
  2. Disrupt tissue by shaking with paint shaker for 2 min. Repeat if clumps of tissue remain.
  3. Centrifuge briefly to bring down tissue dust.
  4. Add 300 ul CTAB, place sealing mat loosely on top of plate and heat at 65°C in water bath for 30 min. (optional)
  5. Let cool to room temperature.
  6. Centrifuge briefly and add 300 ul chloroform (done in hood). Use a TIP BOX lid for chloroform; it melts the boats! Seal tightly with the sealing mat and vortex vigorously for 10-20 seconds. Or use the mix step on the Apricot.
  7. Centrifuge at 3250 rpm for 15 min.
  8. During centrifugation, prepare 96-well deep well plates by adding 200 ul isopropyl alcohol to each well.
  9. Transfer 200 μl of the chloroform-extracted supernatant to the new plates. Be very careful not to transfer any of the goop from the interface, or any of the organic layer.
  10. Cover the tubes with the sealing mat and centrifuge at 3250 rpm for 15 min.
  11. Pour off the liquid into the sink; the pellet of DNA should stay behind.
  12. Wash with 200 μl of 70% ethanol, re-cover the tubes and centrifuge for 7-10 min. at 3250 rpm.
  13. Dump off the liquid again and blot the plates dry on paper towels. Cover the plates with a kimwipe and let them air dry for >3 hours (or until no liquid can be seen inside and the tubes don’t smell like ethanol).
  14. Resuspend the pellets in 100 ul of TE and let sit for >3 hours at 4°C (I usually leave them overnight). The DNA can then be transferred to skirted 96-well PCR plates for storage.
  15. Use 0.5 μl in a 10 μl PCR reaction.

 

1 Liter of 2X CTAB


 

  • 20g CTAB (cetyltrimethyl or hexyltrimethyl ammonium bromide)
  • 81.82g NaCl
  • 100 ml 1M Tris, pH 8
  • 40 ml 0.5M EDTA
  • (CTAB goes into solution slowly and can release toxic fumes if heated)
  • Final cc:
  • 2% CTAB, 1.4M NaCl, 100 mM Tris, 20mM EDTA
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