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now: Wed Jan 27 23:36:30 2021 ... mod: Mon Jul 1 21:17:42 2013
That's more than snesilbe! That's a great post!
PHENOL-FREE GENOMIC DNA PROTOCOL
For isolating DNA from tissue culture cells. This protocol yields "high molecular weight" genomic DNA with an average size of about 50 kbp, and generally larger than 30 kbp. There is almost no DNA of 100 kbp or larger size produced -- use the HighMwGenomicDna
protocol if very large DNA fragments are required.
- For adherent cells, rinse with PBS and disperse with trypsin/EDTA.
- for typical monolayer mammalian cells, a semi-confluent 10 cm plate will yield enough genomic DNA for extensive SouthernBlotting analysis
- Pellet cells and remove supernatant.
- Resuspend the cell pellet in whatever residual liquid remains after removal of the supernatant in the above step. Generate a homogenous cell suspension by alternately flicking the bottom of the tube and/or vortexing.
- getting the pellet loosed up before adding the SALT-X solution in the next step will greatly help thorough digestion
- Add 400μl SALT-X solution to cells. Mix by gentle agitation. Incubate at 50C until solution clears.
- proteinase K has high activity over a broad temperature range of from 37C to 60C. proteinase K begins to destabilize around 65C depending upon buffer conditions
- a cleared solution can be transparent to translucent but should look completely homogenous. this can take a few hours to several days depending on number of cells
- agitation (not vortexing) periodically can help disperse cell pellets and clumps
- Remove digested cells to an eppendorf tube. Add 300μl NaCl saturated water and shake tube vigorously. Do not vortex. A white precipitate should form immediately.
- Centrifuge 3 minutes to pellet proteins. If the pellet is not solid, shake vigorously again and repeat this step.
- If the pellet is not tight and has a gluey character, it means either that the cells were not completely digested by the SALT-X solution, or that there was incomplete mixing of the DNA with the saturated salt solution. Vigorous repeat shaking can help in this latter case.
- Remove supernatant to a new tube and recentrifuge.
- Remove 600μl of supernatant to a new tube. Avoid any pellet and/or cloudiness.
- Add 420μl room temperature isopropanol and mix by repeated gentle inversion. Precipitated DNA should be evident. Let sit for 3 minutes.
- Pellet genomic DNA in microfuge at 15000g for 3 minutes. Rinse pellet with room temperature 75% EtOH, aspirate ethanol, let air dry and resuspend in 0.1x TE.
400 mM NaCl
10 mM Tris pH 8.0
2 mM EDTA pH 8.0
0.4 mg/ml proteinase K → added immediately before use
- proteinase K can be stored as aliquoted stocks frozen at -20C at 15 mg/ml in water
- freeze/thawing proteinase K aliquots is not recommended, but likely causes little loss of activity
- Store the SALT-X solution without added proteinase K at room temperature.
NaCl SATURATED WATER
Try to make approx 7M NaCl in water.
Store at room temperature with undissolved salt on the bottom.
Use the liquid phase.
10 mM Tris pH 8.0
1 mM EDTA pH 8.0
For 0.1x, dilute with H2O. Using 0.1x helps subsequent restriction digestion if genomic DNA solution makes up a large proportion of the digest.
Aljanabi and Martinez, Nucleic Acids Research, 25(22): 4692-4693. (1997)
Miller and Polesky, Nucleic Acids Research, 16(3): 1215. (1988)
- This protocol is faster, cheaper and less messy than protocols involving phenol extraction. DNA is suitable for restriction digestion, SouthernBlotting and PCR.
- The isolated DNA is largely RNA-free making an RNase step unnecessary. don't know why, but this does seem to be the case