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 ul 10% SDS, invert / shake the tube a few times. Incubate at 65 C for 15 min.
• Add 300 ul 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 ul of the supernatant to a new eppendorf tube.
• Add 900 ul NaI (GeneClean II), + 20 ul  "glass  milk" (silica particles) to the supernatant, (total volume of 1220 ul). 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 ul wash solution (from the GeneClean II kit).
• Repeat the wash two times.
• Dry the pellet (with bound DNA).
• Resuspend the pellet in 50 ul distilled water. Incubate at 50-65 C 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 ul 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. （）