Rapid Extraction Protocol
USDA-ARS Genotyping Facility
Submitted by Gina Brown-Guedira
This protocol has been submitted for publication as a NOTE in Crop Science
- 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.
- Add 40ul 0.25 NaOH directly into wells containing leaf tissue.
- Heat microtiter plate in 95C water bath for 1 min.
- Put stainless steel cylinders on top of tissue. Using the matrix mill, grind tissue for 7-10 min.
- After grinding remove cylinders. Add 130ul of 0.1 Tris-HCl to each well.
- Spin plate 3500rpm, 10min.
- Remove 150ul supernatant and put into a 200uL 96-well conical bottom PCR plate
- Add 15ul 3M NaOAc and 120ul 100% isopropanol
- Freeze 80C 1hr. or 20C overnight
- Centrifuge 3500rpm, 30min
- Remove isopropanol and centrifuge upside down 600rpm, 1min
- Add 200ul 70% EtOH
- Centrifuge 3500rpm, 20min
- Remove EtOH and centrifuge upside down 600rpm, 1min
- Resuspend in 20ul TE buffer
Products we use:
8-well rectangular tissue culture dish (Nunc, Cat. No. 176600)
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,
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