Quick DNA prep (Wright lab, Nov. 13, 1997)
Trypsinizecountpellet (pellet can be frozen and stored or processed immediately)
Resuspend in 30 µl per million cells in:
100mM NaCl
100 mM EDTA pH 8
10 mM Tris pH 8
AFTER resuspending, add Triton X-100 to 1% from a 25% stock (use Triton rather than
SDS so that you don't have to worry about residual detergent later on--Triton doesn't
inhibit restriction enzymes), then add protease K to 2 mg/ml.
Digest 55C for 2 hours (overnight is OK but unnecessary).
Inactivate protease K 70C for 30 min.
Dialyze overnight in cold room (e.g., small diameter Spectra/Por membranes MWCO
12-14000, but virtually anything should be OK). We have used as few as 500,000 cells
with no problems. After dialysis, an original vol. of 30 µl is still less than 140 µl, so that
even with only 500,000 cells (which is 3 µg) the volumes are still small enough to
analyze without any concentration step. The main purpose of the dialysis is to reduce
the salt and EDTA concentrations so that it doesn't complicate the digestion. One can
establish conditions of simply adding Mg++ to neutralize the EDTA, but this requires
carefully neutralizing all of the acid that is liberated so the pH is right for the digestion.
Dialysis is simpler and more reproducible. Best to dialyze a couple hours, then change
the TE and dialyze overnight.
(January 2000 modification: Using 1.8 ml screw-cap tubes, one can facilitate the
process as follows. Cut off the very top of the screw-cap, so that the threads remain but
the top is gone (using a jigsaw, it is possible to cut off the top of 100 caps in about a half
hour). This enables one to put a small piece of dialysis membrane over the open top of
the tube, then screw the “topless cap” on it. When inverted and placed in a rack (e.g., a
foam rack) so that the membrane is 1 mm or so under the surface of 4L beaker, this
then provides a convenient microdialysis system, so that dozens of samples can be
processed at once and the DNA can be stored in the small tubes in which they are
dialyzed (after dialysis, replace the topless cap with a normal cap). NB: be careful that
all of the DNA is flicked down to lie against the membrane so it can get dialyzed.)
Based on final volume after dialysis, calculate DNA content using 6µg/million normal
cells or 10µg/million aneuploid (tumor) cells. The DNA should be VERY viscous.
Usually, the first time someone tries it they don’t get viscous high MW DNA, but after
they repeat it they do.
Typical sequence:
Monday: Trypprotease K digest and inactivatedialyze overnight
Tuesday: Digest during day, load gel and run overnight
Wednesday: Dry gel, hyb overnight
Thursday: wash, expose.
Advantages:
1) VERY consistent yields based upon cell counts (no variability due to
phenol/chloroform and or precipitation losses etc.)
2) Very fast, easy, and reproducible
3) Can use very small numbers of cells (we have used it for as few as 500,000 to as
many as 60 million cells)
4) Very easy to process large numbers of samples, since one avoids the multiple
transfers from tube to tube of phenol/chloroform extractions. (It can be a one-tube
process if the cells are spun down in the same tube in which they are digested.
Depending on the number of cells, we sometimes spin them in a 0.5 ml tube, then do
the digestion and inactivation in a PCR machine).
TRF analysis (Wright Lab, Nov 13, 1997)
Digest 2 µg in 40-140 µl of Boehringer-Mannheim Buffer A using 2.5 µl (e.g. 25 total
units) of an equal mixture of six enzymes:
All Boehringer-Mannheim, 10U/µl
AluI, CfoI, HaeIII, HinfI, MspI, RsaI
Thus, per enzyme, are using about 2U per microgram DNA. This is probably a HUGE
excess, but we haven't taken the time yet to verify if we can get away with less. We
never get partial digests.
Digest 4 hours or overnight.
Run 0.7% Agarose gel. Stop the gel and take a picture BEFORE small molecular weight
products migrate off the gel to verify complete digestions. We run at very low voltage
overnight, take a picture the next morning, then crank up the voltage for a few hours
until the 1.5 Kb marker is near the bottom of the gel.
Denature 20 min in 0.5M NaOH, 1.5M NaCl
Rinse dH20 and incubate in dH20 for 10 min. (this gets rid of most of the NaOH and
substantially reduces sticking of the gel to the 3mM paper)
Dry one hour 50 C.
Neutralize 10 min in 0.5M tris 8, 1.5M NaCl
Pre-hyb 10 min (mainly to get buffer the same as hyb)
Hyb O.N. 40C with 3-repeats (either G or C strand: G probe for the C-rich strand tends
to give a higher signal intensity for the same input counts)
Wash 2x SSC at 40C for 10-15 min.
Wash 0.1x SSC/0.1% SDS 15 min x 2  expose.