DNA Purification from
0.5-2.0 mg solid tissue (Expected yield range 0.3-1.5 µg
- Dissect tissue sample quickly and freeze in liquid nitrogen.
Store at -70° to -80°C.
Fresh tissue may also be used. Work very quickly and keep tissue on
ice at all times
including when tissue is weighed.
- Add 0.5-2.0 mg (0.0005-0.002 g) frozen ground tissue or fresh
tissue to a 1.5 ml tube
containing 100 µl Cell Lysis Solution, remove from ice, and
using a microfuge tube pestle. Place sample back on ice until next
- Incubate lysate at 65°C for 15 minutes. Alternatively, if
maximum yield is required,
0.5 µl proteinase K Solution (20 mg/ml) may be added to the lysate.
inverting 25 times and incubate at 55°C for 3 hours to overnight,
particulates have dissolved. If possible, invert tube periodically
- Add 0.5 µl RNase A Solution (4 mg/ml) to the cell lysate.
- Mix the sample by inverting the tube 25 times and incubate at
37°C for 15-60
- Cool sample to room temperature.
- Add 33 µl Protein Precipitation Solution to the RNase A-treated
- Vortex vigorously at high speed for 20 seconds to mix the
Solution uniformly with the cell lysate. Place sample on ice for 5
- Centrifuge at 13,000-16,000 x g for 3 minutes. The precipitated
proteins will form a
tight pellet. If the protein pellet is not visible, repeat Step 3
followed by incubation on
ice for 5 minutes, then repeat Step 4.
- Pour the supernatant containing the DNA (leaving behind the
pellet) into a clean 1.5 ml centrifuge tube containing 100 µl 100%
If the DNA yield is expected to be low (<1 µg), add a DNA
carrier such as
Gentra Glycogen Solution (0.5 µl glycogen 20 mg/ml) to the 100 µl
- Mix the sample by inverting gently 50 times.
- Centrifuge at 13,000-16,000 x g for 5 minutes.
- Pour off supernatant and drain tube on clean absorbent paper.
Add 100 µl 70%
Ethanol and invert tube several times to wash the DNA pellet.
- Centrifuge at 13,000-16,000 x g for 1 minute. Carefully pour
off the ethanol. Pellet
may be loose so pour slowly and watch pellet.
- Invert and drain the tube on clean absorbent paper and allow to
air dry 10-15
- Add 20 µl DNA Hydration Solution (20 µl will give a
concentration of 50 ng/µl if
the total yield is 1 µg DNA).
- Rehydrate DNA by incubating sample 1 hour at 65ºC and/or
overnight at room
temperature. If possible, tap tube periodically to aid in
dispersing the DNA.
- Store DNA at 4°C. For long-term storage, store at -20°C or
Copyright 2003 Gentra Systems, Inc.
Printed in USA •
2/03• 00440 Rev B
puregene_dna_isolation_from_ears.pdf - Added on May 24, 2010 at 9:41 PM by Jill Lindner