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Rapid Extraction Protocol

USDA-ARS Genotyping Facility

Manhattan, KS

Submitted by Gina Brown-Guedira

This protocol has been submitted for publication as a NOTE in Crop Science

  1. Add coleoptiles or leaf tissue directly into a 96-well flat bottom tissue culture plate. Roll up tissue to fit into well, do not let tissue stick to the side of the plate. Freeze tissue at –20C until ready to extract. **Fresh tissue works best.
  2. Add 40ul 0.25 NaOH directly into wells containing leaf tissue.
  3. Heat microtiter plate in 95C water bath for 1 min.
  4. Put stainless steel cylinders on top of tissue. Using the matrix mill, grind tissue for 7-10 min.
  5. After grinding remove cylinders. Add 130ul of 0.1 Tris-HCl to each well.
  6. Spin plate 3500rpm, 10min.
  7. Remove 150ul supernatant and put into a 200uL 96-well conical bottom PCR plate
  8. Add 15ul 3M NaOAc and 120ul 100% isopropanol
  9. Freeze –80C 1hr. or –20C overnight
  10. Centrifuge 3500rpm, 30min
  11. Remove isopropanol and centrifuge upside down 600rpm, 1min
  12. Add 200ul 70% EtOH
  13. Centrifuge 3500rpm, 20min
  14. Remove EtOH and centrifuge upside down 600rpm, 1min
  15. Resuspend in 20ul TE buffer

Products we use:
    Germination containers
    8-well rectangular tissue culture dish (Nunc, Cat. No. 176600)

Extraction plastics:
    96-well flat bottom tissue culture dish (Nunc, Cat No. 167008)
    Conical bottom 96-well PCR plate (Genemate®, Cat no. T-3031-21)

Mention of trademark or proprietary product does not constitute a guarantee or warranty by the USDA and does not imply its approval over other suitable products.

Questions? Contact Dr. Kristi Hill-Ambroz, e-mail: wheatgenes@yahoo.com

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