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 .
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