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 m l of preheated (65°C) extraction buffer, mix and let float in waterbath at 65°C for 10 to 60 min
5. spin down 15 to 20 min (5000 rpm) and transfer 300 m l of the supernatant to a new 96 well plate (E&K, 1ml, polypropylene) containing 300 m l 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 m l 1x TE
10. use 1-3 m l per PCR rxn

 

Solutions:

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