Genomic DNA Extraction for Mapping

1.grow plants in trays of 96 and leave two spots open (for the PCR controls)

2.harvest 1 to 2 young and green leaves (1cm2 /plant, at rosette stage if possible). Use 96 well plates (1 or 2 ml, E&K, polypropylene, round bottom). Process all and freeze in liquid nitrogen

3.grind tissue for 30 sec per row with a "12-finger comb" (specially built to fit into the wells) while still frozen

4.add 400 μl of preheated (65℃) extraction buffer, mix and let float in waterbath at 65℃ for 10 to 60 min

5.spin down 15 to 20 min (5000 rpm) and transfer 300 μ l of the supernatant to a new 96 well plate (E&K, 1ml, polypropylene) containing 300 μl isopropanol/well

6.mix and wait 5 min at RT (can wait much longer)

7.spin 30 to 45 min (5000 rpm). Pour off supernatant

8.wash pellets with 70% EtOH and air dry (e.g., ovn)

9.resuspend pellets in 50-100 μl 1x TE

10.use 1-3 μl per PCR rxn

Solutions:

Extraction Buffer
1 M Tris-HCl pH 7.5	10.00 ml
5 M NaCl	2.50 ml
0.5 M EDTA	2.50 ml
20% SDS	1.25 ml
H2 O	33.75 ml
Total	50.00 ml

Remarks:

This protocol is quick, reliable and the DNA obtained can be used for PCR-based mapping using SSLP and CAPS markers. It is based on the protocol by Edwards et.al., 1991, NAR 19: 1349. It was adapted for large-scale applications by the Somerville lab .