Protocol Online: Cached

This is a cached page for the URL (http://boneslab.chembio.ntnu.no/QuickPrepDNA.html). To see the most recent version of this page, please click here.

Protocol Online is not affiliated with the authors of this page nor responsible for its content. About Cache

Quick Prep DNA

A quick method to isolate plant genomic DNA.

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.

Samme prosedyre men på Norsk: (Beklager men ikke på ny-Norsk)

Vei opp 0.1 gram av plante materiale.
Knus materiale i morter, med alumina og flytende N2.
Overfør pulver til 1.5 ml eppendorfrør.
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 130 ul 10% SDS, bland godt, og inkuber ved 65 C i 15 min.
Tilsett 300 ul 5M kalium acetat. Bland godt. La løsningen stå på is i 30 min.
Spinn ned ved 7000 rpm, 5 min. og ta ut 300 ul av supernatant.
Til de 300 ul med supernatant tilsettes 900 ul NaI (GeneClean II), + 20 ul "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 løsningen vil nå være bundet til silika partiklene).
Vask silika pellet med 800 ul vaskeløsning (GeneClean II).
Gjenta vasketrinn 2 ganger.
Tørk silika pellet ved 65 C, ca 10 min.
Resuspender pellet i 50 ul destilleret vann. Inkuber ved 50-65 C i 5 min.
Spinn ned pellet og ta av supernatant som inneholder DNA.