Demo DNA

Rosnthorn at aol.com Rosnthorn at aol.com
Mon Aug 19 20:44:38 EST 1996


This is how I showed EPA (TSCA division of biotechnology) and the Smithsonian
(Museum of Natural History Laboratory of Molecular Systematics) to prepare
DNA for demonstrations to the public.  Properly cared for, the pre-ethanol
aqueous solution lasts at least three years (to date) by simply handling
aseptically when removing samples and storing the remainder in the
refrigerator.  In this method, no phenol or chloroform or protease are
required. Do not shake the material hard or vortex it (except when first
resuspending the cells in PBS) or pipette up and down a lot.  Pouring works
fine and keeps the DNA long so fibers form easily

1. Grow up a liter of E.coli (non-plasmid bearing). 
2. Spin the cells at 2-5K for 10 minutes
3. Pour off the supernatant
4. Resuspend the pellet in 500 ml of Phosphate-buffered saline (from your
e-mail address it seems you have access) It is extremely important to have
the saline.  Even 0.15 M is sufficient to neutralize the DNA so it can be
precipitated later 
5. Add about 10 ml of 0.25-0.5 M EDTA (a good preservative at this high
concentration)
6. Add several drops of Joy detergent or about 5 ml of 10-20% SDS
7. Swirl carefully and begin heating at 65C
If the solution doesn't begin to clear in 15 min (by the time the entire
solution is warmed), add a few more drops (or ml) of detergent and return to
65C
Due to the high concentration of cells, you will probably need to incubate a
good 2 h or so, and I have kept the prep at 65C overnight
8. Allow to cool to room temp and then refrigerate to store for extended
periods
9. To precipitate the DNA, POUR a small amount (a few ml) into a 50 ml tube
or beaker or pipette 2ml into a 10 ml tube.  The solution will be clear, but
obviously viscous
10. Add 2 vol of cold 95% EtOH (NOT isopropanol and NOT 70%EtOH) carefully
down the side of the tube so the 2 liquid layers stay separate
11. The DNA will come out of solution and trap large bubbles as the fibers
form.
You can store the large volume of solubilized cells in the refrigerator
indefinitely.  A white precipitate (protein??) may form eventually.  I try
not to stir the liquid and to simply pour the needed quantity so the ppt does
not carry over
You can add 2 vol of room temperature 95% EtOH to the chilled aqueous
material and the layers will remain immiscible.
Regards,
Toby Mogollon Horn, Ph.D.   *the DNAHAND*   thorn at lan.tjhsst.edu
Thomas Jefferson High School for Science and Technology
Life Science and Biotechnology Laboratory
Alexandria, VA 22312     703-750-5024



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