(adapted from McKinney et al., 1995, Siebert, 1995, Strauss et al., 2001 and Alonso et al., 2003)
PURPOSE: To analyze unknown flanking genomic sequences adjacent to a T-DNA left border
1.) Isolation of DNA
-collect 2-3 young leaves in an eppendorf tube
-add 100 uL extraction buffer and add proteinase K, grind tissue using a blue pestel (no large pieces of leaf should be left).
-add another 100 uL of extraction buffer, vortex, and incubate in 37C for 30 min.
-add 200 uL of saturated phenol and vortex.
-spin at max speed in centrifuge for 2 min.
-collect upper phase to new eppendorf tube.
-add 200 uL of (24:1) chloroform:isoamyl alcohol, vortex, centrifuge at max speed for 2 min.
-collect upper phase into a new eppendorf tube.
-add 18 uL of 3M sodium acetate and add 400 uL of 100% EtOH, mix by inverting and incubate for 10 min at 4C.
-spin in centrifuge at max speed for 10 min.
-pour supernatant off and wash with 500 uL of 70% EtOH
-spin in centrifuge at max speed for 5 min.
-pour supernatant off and wash again with 500 uL of 70% EtOH
-spin in centrifuge at max speed for 5 min.
-pour off supernatant
-carefully pipette off excess EtOH
-let pellet dry for 45 min in the hood.
-resuspend DNA in 100 uL of TE
-store in -20C