X - Services
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.
- Degenerate PCR
- Quick prep - DNA isolation