Plant DNA mini-prep for PCR
(adapted from McKinney et. al. 1995)
1. Collect 1-2 small young leaf (0.5 cm wide) in an eppendorf tube
2. Add 100 ml of extraction buffer and grind the leaf using a blue plastic pestle, leaves should be
ground till there are no large bits of leaf left intact.
3. After all samples are ground add an additional 100 ml of extraction buffer, vortex the samples
briefly, and incubate at 37°C for 30 minutes.
4. Add 200 ml of buffer saturated phenol to each sample, vortex, and spin in the microfuge at
maximum speed for 2 minutes.
5. Transfer the top aqueous layer to a new tube.
6. Add 200 ml of chloroform / isoamyl alcohol (24:1), vortex and spin in the microfuge at
maximum speed for 2 minutes.
7. Transfer the top aqueous layer (being sure to not take up any of the chloroform) to a new tube.
8. Add 18 ml of 3M sodium acetate and precipitate the nucleic acids by adding 400 ml of ( 2
volumes) 100 % ethanol.
9. Pellet the precipitated DNA by spinning in the microfuge for 10 minutes at maximum speed.
10. Pour off the supernatant being careful not to loose the pellet.
11. Wash the pellet twice with ~500 ml of 70% ethanol and air dry the pellet.
12. Resuspend the DNA in 100 ml of T.E.
13. Store in 4 °C.
EXTRACTION BUFFER
50mM Tris pH 8.0
200 mM NaCl
0.2 mM EDTA
0.5% SDS
100 mg/ml Proteinase K
100 mg/ml glycogen (optional)
100 ml stock
2.5 ml of 2M
4.0 ml of 5M
40 µl of 0.5M
5 ml of 10%
*
*
* for a working solution add 50 ul of 20 mg/ml to 10 ml of stock just before use.