TISSUE DIGESTION
1. Prepare enough 1.5 ml tubes. Use the Flat Top Fisher tubes (light
pink) only. Other 1.5 mL tubes leak. Get working stock solutions from the
-20oC "DNA extractions" rack" and use the common Extraction
Buffer stock
Normally use:
2. Obtain sterile razor blades and kimwipes. Remove about
10-30 ul of tissue per sample. Chop the tissue up with the razor
blade. Optional: you may want to use a blue plastic pestle
to grind the tissue in the tube.
3. Incubate at 50-60oC on a rocking tray overnight
or at least for 3 hours.
(the tissue should be digested to completion)
PHENOL EXTRACTION AND ETHANOL PRECIPITATION OF DNA
This protocol describes the most commonly used method of purifying and concentrating DNA preparations. The DNA solution is first extracted with a phenol/chloroform/isoamyl alcohol mixture to remove protein contaminants, then precipitated with 100% ethanol. The DNA is pelleted after the precipitation step, washed with 70% ethanol to remove salts and small organic molecules, and resuspended in buffer at a concentration suitable for further experimentation.
1. Add an equal volume of PCI (phenol/chloroform/isoamyl alcohol) to the DNA solution to be purified in a 1.5-ml microcentrifuge tube.
Extracting volumes <100 ul is difficult; small volumes should be
diluted to obtain a volume that is easy to work with.
High salt concentrations can cause the inversion of the aqueous and
organic phases. If this happens, the organic phase can be identified by
its yellow color.
2. Mix gently for for 5 min (rocking platform) and microcentrifuge 10 min at 10,000 rpm at room temperature.
Phases should be well separated. If DNA solution is viscous or contains a large amount of protein, it should be spun longer.
3. Carefully remove the top (aqueous) phase containing the DNA using a 1000-ul pipetor and transfer to a new tube. Repeat steps 1-3. If after repeating a white precipitate is present at the aqueous/organic interface, repeat steps 1 to 3 once again.
4. Add an equal vol of CI (chloroform:isoamyl alcohol 24:1). Mix gently for 2 min and spin for 1 min at 10,000.
5. Carefully remove the top (aqueous) phase containing the DNA using a 1000-ul pipettor and transfer to a new tube.
6. Add 2 to 2.5 vol of ice-cold 100% ethanol. Mix gently and place in -20 C overnight or in -70oC for 1 hr.
7. Spin 20 min in a fixed-angle microcentrifuge at maximum speed and remove the supernatant.
For large pellets the supernatant can simply be poured off. For small pellets (<1 ug), aspirate off the ethanol supernatant with a pipetting device such as a Pasteur pipet or pipettor. This is best accomplished by drawing off liquid from the side of the tube opposite that against which the DNA precipitate was pelleted. Start at the top and move downward as the liquid level drops.
7. Carefully add 1 ml of room-temperature 70% ethanol. Invert the tube gently and microcentrifuge as in step 7.
If the DNA molecules being precipitated are very small (<200 bases), use 95% ethanol at this step.
8. Remove the supernatant as in step 5. Dry the pellet at room temp.
The DNA pellet will not stick well to the walls of the tube after the 70% ethanol wash and care must be taken to avoid aspirating the pellet out of the tube.
9. Dissolve in 20-50 ul TE buffer, pH 8.0.
DNA pellets will not dissolve well in high-salt buffers. To facilitate resuspension, the DNA concentration of the final solution should be kept at <1 mg/ml.
If DNA is resuspended in a volume of TE buffer or water to yield a DNA concentration
of <1 mg/ml, small quantities (<25 ug) of precipitated plasmids or restriction
fragments should dissolve quickly upon gentle vortexing or flicking of the tube.
However, larger quantities of DNA may require vortexing and brief heating (5
min at 65oC) to resuspend. High-molecular-weight genomic DNA may
require one to several days to dissolve and should be shaken gently (not vortexed)
to avoid shearing, particularly if it is to be used for cosmid cloning or other
applications requiring high-molecular-weight DNA. Gentle shaking on a rotating
platform or a rocking apparatus is recommended.
288 ml ddH2O
3 ml 1 M Tris pH 8.0
6 ml 0.5 M Na Cl
... autoclave
then add 2 g solid SDS