植物基因组的快速提取

Use from 0.01 - 0.1 gram plant material.

Grind the plant material with liq. N₂ in a mortar. We normally use some alumina to crush hard tissue.

Transfer the ground tissue to a 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 ℃ for 15 min.

Add 300 μl 5M potassium acetate. Mix well. Keep the solution on ice for i 30 min, (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 room temp. for 5 min.

Spin down the silica particles (glass milk), 5 sec., 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 ℃ for 5 minutes.

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 a agarose gel. If you use 0.1 gram plant material you should be able to see the DNA on the gel.