Arabidopsis thaliana DNA miniprep

(adapted from Dellaporta et al. 1983 Plant Molec. Biol. Reporter 1: 19-21)

1. Prepare tissue (1 leaf or up to 0.4 g) by grinding under liquid nitrogen. Store at -80o C until needed.

2. Set water bath to 65o C. Add 750 ul extraction buffer to frozen leaf tissue. Mix well by vigorous shaking.

3. Add 50 ul 20% SDS, invert several times to mix.

4. Incubate at 65o C for 10 min

5. Add 250 ul 5M KOAC. Invert several times to mix. Incubate on ice 20 minutes.

6. Spin 20 minutes in tabletop microfuge. Transfer supernatant (900 ul) to a clean tube containing 0.6 volumes isopropanol (540 ul).

7. Mix by inversion; incubate 30 min to overnight at -20o C.

8. Pellet DNA in microfuge 10 min. Pour off supernatant and dry pellet in speed vac.

9. Dissolve pellets in 0.4 ml Tris EDTA (50mM, 10mM, pH 8.0). 65o C bath acceptable if needed.

10. Spin 10 min to pellet contaminants. Transfer cleared supernatent to fresh tube, add 1/10th volume 3M NaOAC (pH 5.0) and 2 volumes 95% ethanol. Mix well by inversion, spin 3 minutes in microfuge.

11. Wash pellet in ice cold 70% EtOH, spin 30 sec, remove supernatent.

12. Dry pellet in speed-vac. Dissolve DNA in 10-100 ul TE (10 mM,1mM; pH 8.0).

13. Quantify DNA concentration using spectrophotometer or fluorometer.

 

Extraction Buffer

100 mM Tris pH 8.0
50 mM EDTA pH 8.0
500 mM NaCl
10 mM 2-mercaptoethanol

 

 

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