Extract Genomic DNA from Arabidopsis Leaves (Can be Used for Other Tissues as Well)   

Abstract

Materials and Reagents

1. Ethanol (EtOH)
2. Isopropanol
3. NaCl
4. EDTA
5. SDS
6. Tris (pH 7.5)
7. Edward buffer (see Recipes)
   

Equipment

1. Ceramic mortar and pestles
2. Centrifuges (Eppendorf)
3. Vortex (VWR International)
   

Procedure

1. Add 400 μl Edward extraction buffer to a 1.5 μl tube.
2. Take 2-3 small leaves or 1 big leaf, grind with pestle (2-3 weeks old).
3. Vortex 5 sec, set at room temperature until all preps are ready.
4. Spin at 16,000 rpm for 2 min.
5. Transfer 300 μl of suspension to a fresh tube.
6. Add 300 μl of isopropanol at room temperature for 2 min.
7. Spin 5 min, wash pellet with 70% EtOH, and dry at room temperature.
8. Resuspend in 100 μl H₂O and store at -20 °C.
9. Use 2 - 4 μl for PCR reaction to validate the presence of target DNA.
   

Recipes

1. Edward buffer
   200 mM Tris (pH 7.5)
   250 mM NaCl
   25 mM EDTA
   0.5% SDS
   

References

1. Lu, Y., Chanroj, S., Zulkifli, L., Johnson, M. A., Uozumi, N., Cheung, A. and Sze, H. (2011). Pollen tubes lacking a pair of K⁺ transporters fail to target ovules in Arabidopsis. Plant Cell 23(1): 81-93.