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 2ml, E&K, polypropylene, round bottom). Process all and freeze in liquid nitrogen

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

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

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

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

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

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

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

10.use 1-3 ml per PCR rxn

Solutions:

1.Extraction buffer: 200mM Tris-HCl pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS

2.Extraction Buffer  1M Tris-HCl pH 7.5 10.00ml 5M NaCl 2.50 ml 0.5 M EDTA 2.50ml 20% SDS 1.25ml H₂O 33.75ml Total 50.00 ml