1.Collect leaf tissue from 1 seedtray of 4 week old plants = 30 g tissue = approx 1 Falcon tube full ground tissue.
2.Grind to a fine powder in liquid nitrogen with a mortar and pestle.
3.Place powder into a 250 ml centrifuge bottle and add 50 ml of 2xCTAB buffer pre-heated to 95°C. Mix thoroughly and transfer to a 65°C waterbath.
4.Incubate for 45 mins, mixing occasionally.
5.Allow tubes to cool and add an equal volume of chloroform: isoamyl alcohol (24:1)
6.Emulsify by gentle shaking and centrifuge for 12 mins in benchtop Sorvall.
7.Remove upper aqueous phase to a new tube and add 1/10th vol 10% CTAB pre-heated in 65°C waterbath.
8.Mix thoroughly and repeat chloroform: isoamyl alcohol extraction.
9.To new aqueous phase add 2 x vol CTAB precipitation buffer, mix thoroughly and allow to stand at room temp for 1 hour. This step precipitates CTAB/DNA complexes by reducing NaC1 concentration, so if no precipitate forms, add a little more precipitation buffer. Precipitates may be stringy or fine suspensions.
10.Spin down precipitates for 15 mins in benchtop Sorvall.
11.Drain tubes well and resuspend pellet in 10ml 1M NaC1 and transfer to a Falcon tube.
12.Add two volumes of ethanol to precipitate nucleic acids. Spin down or spool out DNA and rinse twice for 2 mins in 70% ethanol.
13.Finally spin down nucleic acids for 5 mins in benchtop Sorvall, remove all 70% ethanol and vacuum dry pellets for 5 mins.
14.Resuspend DNA in 500µl TE. Check by running 1µl on 0.7% agarose gel.
2xCTAB: 100mM Tris-HC1 pH 8.0, 1.4 M NaC1, 20 mM EDTA, 2% CTAB.
10% CTAB: 10% CTAB, 0.7 M NaC1
CTAB precipitation buffer: 50 mM Tris-HC1 pH 8.0, 10 mM EDTA, 1% CTAB
{CTAB = cetyltrimethylammonium bromide}
NB During the extraction procedure, samples should never be allowed to fall below 15°C, especially during centrifugation, since below this temperature, CTAB precipitates from solution.