Aquickmethodtoisolateplant

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 130μl 10% SDS,invert / shake the tube a few times.Incubate at 65℃ for 15 min.

Add 300μl 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 300μl of the supernatant to a new eppendorf tube.

Add 900μl NaI (GeneClean II), 20μl "glass milk" (silica particles)to the supernatant,(total volume of 1220μl).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 800μl wash solution (from the GeneClean II kit).

Repeat the wash two times.

Dry the pellet (with bound DNA).

Resuspend the pellet in 50μl 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 10μl 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.

Samme prosedyre men p? Norsk: (Beklager men ikke p? ny-Norsk)

Vei opp 0.1 gram av plante materiale.

Knus materiale i morter,med alumina og flytende N₂ .

Overf?r pulver til 1.5 ml eppendorfr?r.

Tilsett 1 ml ekstraksjonsbuffer (100 mM Tris-HCl pH 8.0,50 mM EDTA pH 8.0,500 mM NaCl, 0.07 % 2-mercaptoetanol).Resuspender godt.

Tilsett 130μl 10% SDS,bland godt,og inkuber ved 65℃ i 15 min.

Tilsett 300μl 5M kalium acetat.Bland godt.La l?sningen st? p? is i 30 min.

Spinn ned ved 7000 rpm,5 min.og ta ut 300μl av supernatant.

Til de 300μl med supernatant tilsettes 900μl NaI (GeneClean II), 20μl "glass milk" (silika partikler).Bland godt,inkuber ved rom temp.ca 5 min.

Spinn ned silika partiklene,5 sek.,og ta av supernatant.(DNA i l?sningen vil n? v?re bundet til silika partiklene).

Vask silika pellet med 800μl vaskel?sning (GeneClean II).

Gjenta vasketrinn 2 ganger.

T?rk silika pellet ved 65℃,ca 10 min.

Resuspender pellet i 50μl destilleret vann.Inkuber ved 50-65℃ i 5 min.

Spinn ned pellet og ta av supernatant som inneholder DNA.