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The procedure is suitable for all types of tissues from wide variety of animal, blood
and plant species. All DNA extraction steps are performed at weak acid pH (HEPES
or MOPS free acids) and optionally with hot chloroform for 'difficult' samples, and at
room temperature.
The following protocol is designed for small and large tissue samples (tissue volume
100-200 μl).
Note that isolating genomic DNA not requires gentle mixing because the DNA not be
sheared by vortexing.
1. 2 ml Eppendorf Safe-Lock tube with tissue sample and glass boll freeze at
-80°C, grind in the MM300 Mixer Mill for 3 min at 30 Hz.
2. In 2 ml Eppendorf Safe-Lock tube with mechanically disrupted seeds or leaves
or herbarium add fresh 1 ml of extraction buffer (1% SDS, 3 mM CaCl2, 10
mM EDTA, 0.1 M HEPES, pH 5.3) with 200 μg of proteinase K, vortex very
well and incubate the samples at 60°C for 30-60 minutes.
3. An additional isolation step may be required for samples with high content of
polysaccharides or extracellular material, remove insoluble material from the
extract by centrifugation at maximum speed on table microcentrifuge for 5
minutes. Transfer the cleared solution to a fresh tube.
4. Add 0.3 ml 3 M K-acetate (pH 4.8) to the lysate, vortex very well, and
incubate for 5 min on ice; centrifuge the tube for 10 minutes at 14000 rpm,
4°C.
5. Optional: transferred the upper aqueous layer to a new 2 ml microcentrifuge
tube which contains 0.6 ml of phenol-chloroform and vortex well (30 sec with
max speed). Spin at maximum speed in a microcentrifuge for 3 minutes, RT.
6. Transfer the supernatant into a new 2 ml tube containing 0.5 ml of
chlorophorm and vortex very well (30 sec at maximal speed). Centrifuge the
tubes for 2 minutes at 14000 rpm.
7. Repeat the DNA extraction with chloroform until there will be no interphase.
8. Transfer the supernatant into a new 2 ml tube containing 0.8 ml of 2-propanol,
mix very well, and immediately centrifuge the tubes for 2 minutes at 14000
rpm, RT.
9. Discard the supernatant and add 1.5 ml of 70% ethanol into tube and vortex
well; centrifuge the tube for 2 minutes at 14000 rpm and again discard the
supernatant.
10. Do not dry DNA pellet and dissolved immediately in 300 µl of 1xTE, pH 8.0
(with RNAse A) at 55°C for 10-20 minutes.