You are here: > Front page > Methods


Front page
Staff
Research
Links
Methods
Publications

FUGE NARC
 X - Services

Open Positions
A Rapid Method to Isolate Plant Genomic DNA

  • Use from 0.01 - 0.1 g plant material.
  • Grind the plant material with liquid N2 in a mortar. We normally use some alumina to crush hard tissue.
  • Transfer the ground tissue to an eppendorf tube.
  • Add 1 mL extraction buffer (100 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0, 500 mM NaCl + 0.07% 2-mercaptoethanol). Mix well.
  • Add 130 µL 10% SDS, invert/shake the tube a few times. Incubate at 65°C for 15 min.
  • Add 300 µL 5M potassium acetate. Mix well. Keep the solution on ice for 30 min (allows for precipitation of proteins).
  • Spin down the precipitations at 7000 rpm, 5 min. Transfer 300 µL of the supernatant to a new eppendorf tube.
  • Add 900 µL NaI (GeneClean II), + 20 µL "glass milk" (silica particles) to the supernatant, (total volume of 1220 µL). Mix well and incubate at RT for 5 min.
  • Spin down the silica particles for 5 s, remove supernatant (the DNA in the solution will now hopefully be bound to the silica particles).
  • Wash the silica pellet with 800 µL wash solution (from the GeneClean II kit).
  • Repeat the wash two times.
  • Dry the pellet (with bound DNA).
  • Resuspend the pellet in 50 µL distilled water. Incubate at 50-65°C for 5 min.
  • Spin down pellet and transfer the eluted DNA to a new eppendorf tube.

At this point you should have enough DNA to run 10-20 PCR reactions.

Optional: You can check 10 µL of the eluate on an agarose gel. If you use 0.1 gram plant material you should be able to see the DNA on the gel.





Methods/protocols:

- Degenerate PCR
- Quick prep - DNA isolation