Annotated and modified by
Maitreya Dunham and Cheryl Christianson (November 2005)
Before you start, make the
lysis buffer and TE+RNase, weigh out the glass beads into individual servings,
and label all your tubes. Process
only the number of tubes you can fit in your vortexer at one time (for us
that's batches of 8). Use
phenol-resistant gloves.
Grow an overnight culture
in 5 ml YPD (or ~108-109 cells however you like).
Spin to pellet. Decant super. Resuspend pellet in 500 ml water and transfer to a 1.5 ml screwtop or
lidlock tube. This precaution is
to limit the chance of the tube popping open and spraying phenol everywhere
during the later vortex step.
If desired, the cells can
be stored for later processing by resuspending the pellet in sorbitol solution
(recipe below) instead of water and storing at Ð80C. Upon thawing, proceed as usual.
Spin to pellet again. Decant most of the super, leaving just
enough to resuspend the pellet completely.
To resuspended pellet, add:
|
200 ml |
lysis buffer (recipe
below) |
|
200 ml |
25:24:1
phenol/chloroform/isoamyl alcohol (isoamyl alcohol optional) |
|
300 mg |
acid-washed glass beads
(~500 micron size range) |
Vortex 8 minutes. We've found this added vortexing
increases the yield substantially without obviously shortening the DNA on a 1%
gel. We use a small foam multi-tube attachment, ~4 inches in diameter. Do not use one of those funny rack
vortexers or a large multitube attachment on a normal vortexer. You should make sure your setup actually
vortexes the tubes adequately. If
you get low yields, this is a key step to check.
Touch spin in a low speed
minifuge to get the phenol off the lid.
Add 200 ml TE.
Invert to mix.
Spin 5 min max speed in a
microcentrifuge.
Carefully transfer aqueous
(top) layer to a new tube without catching interphase junk.
Add 1 ml room temp 100%
ethanol. Invert to mix.
Spin 2 min max speed.
Decant super and resuspend
pellet in 400 ml TE+30 mg RNaseA.
The pellet may not resuspend easily. As the incubation proceeds, you can usually get the whole
thing to resuspend, though.
Incubate 30 minutes at
37C. We've lengthened this
digestion from the original 5 min to reduce RNA contamination and to make sure
the entire pellet gets into solution.
Add 10 ml 4 M ammonium acetate and 1 ml room temp 100%
ethanol.
Invert to mix.
Spin 2 min max speed.
Decant super completely
and dry pellet. We leave the tube
inverted on a kimwipe on the bench for about 10 min.
Resuspend in 50 ml TE.
Measure DNA concentration
using a fluorometer or other DNA-specific method (i.e., NOT the
spectrophotometer). Even with the
RNase treatment and ammonium acetate precipitation, there's a lot of RNA
contamination in these preps.
Total yield should be
10-20 mg. DNA
should restriction digest easily.
Lysis buffer
We make this fresh each
time for some unknown reason.
2% Triton X-100
1% SDS
100 mM NaCl
10 mM Tris pH 8
1 mM EDTA
0.9 M sorbitol
100 mM Tris pH 8
100 mM EDTA