To obtain a more pure DNA preparation from pollen the extraction medium was supplemented with reagents capable of suppressing the precipitation of starch.
CTAB extraction buffer: 1% CTAB, 1M Tris-7.5, 5M NaCl, 1M EDTA-8.0, 0.001 mg/ml protease K, dH20.
SDS extraction buffer: 20% SDS, 1M Tris-8.0, 5M NaCl, 1M EDTA-8.0, 0.001 mg/ml protease K, dH20.
For 1000mg of pollen 10 ml of extraction buffer was used. Isolation was done using the following procedure.
1. Grind 1000 mg of fresh pollen grains with a mortar and pestle using liquid nitrogen, into fine powder.
2. Immediately after all the liquid nitrogen has evaporated add 5 ml of the extraction buffer and continue to grind and transfer into a 15 ml polypropylene tube. Add 5 ml of the extraction buffer to the mortar and transfer the remaining ground pollen into the polypropylene tube. Vortex briefly, or mix several times by inversion.
3. Incubate for 60 min with continuous gentle rocking at 60 C.
4. Remove tubes from oven, wait 4-5 minutes and add 4.5 ml phenol/chloroform/isoamyl (25:24:1). Rock gently to mix for 5 min.
5. Spin in table top centrifuge for 10 min.
6. Pour off the top aqueous layer into 15 ml polypropyl-ene tubes. Add 4.5 ml chloroform/isoamyl (24:1) and rock gently for 5 min.
7. Spin in table top centrifuge for 10 min.
8. Pipette off top aqueous layer into 15 ml polypropyl-ene tubes. Add 1.8 ml of NaCl and 1.5 ml of CTAB/NaCl solution and incubate at 65 C for 20-30 min.
9. Add 4.5 ml of chloroform/isoamyl and rock gently for 5 min.
10. Pipette off the top aqueous layer and add 5 ml of NH4OAc. Put the 50 ml polypropylene tube containing the aqueous solution at -80 C for 10 min and then add 25 ml of cold EtOH. Mix by gentle rocking.
11. Remove nucleic acids with a glass rod (or centrifuge for 10 min and then decant and vacuum dry for 1-2 hrs) and dissolve in 500 ul of T.E. (10 mM Tris-8.0, 1 mM EDTA-8.0).
To get rid of the RNA and obtain only
DNA the aqueous solution (in step 10) was treated with RNase A and RNase
T1 for about 30 min before precipitation with EtOH. Approximately 0.4 ug/ul
of DNA was obtained by these methods. Care was taken to isolate nucleic
acids rapidly and to store the samples at 4 C to avoid denaturing of DNA.
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