Large scale P1 prep
Daniel.L.Burgess at um.cc.umich.edu
Thu Dec 8 11:44:44 EST 1994
Several people have been looking for an efficient method for prepping
large amounts of P1 clone DNA. This protocol provides an average of 1
mg of high mol wt. DNA per 1 L culture:
Large Scale P1 Prep:
1) Innoculate a single P1 colony into 30 ml LB medium (25 ug/ml kanamycin)
and incubate overnight with shaking at 37¡C.
2) Next morning, split the overnight culture into each of two 2 L flasks
containing 500 ml LB (25 ug/ml kanamycin). Incubate with shaking at
37¡C for 1.5 hrs.
3) Add IPTG to a final concentration of 0.5 mM. Continue growth for an
additional 5 to 7 hrs. Longer (or overnight) growth lowers yield and
may induce deletions.
4) Transfer 500 ml of culture to two 250 ml centrifuge bottles and chill
on ice for 10 min (store remaining 500 ml at 4¡C temporarily).
5) Centrifuge the two bottles 20 min, 4K rpm, at 4¡C. Pour off and
6) To each bottle (with pellet) add another 250 ml of remaining culture
(was at 4¡C) and centrifuge again 20 min, 4K rpm, at 4¡C. Pour off
as much supernatant as possible.
7) Resuspend* each pellet in 5 ml of ice cold:
50 mM glucose
10 mM EDTA
25 mM Tris-HCl (pH8.0)
*(Use a wide-bore pipette if necessary and resuspend gently to prevent
contamination of P1 with E.coli genomic DNA)
8) Transfer each suspension to a seperate Oakridge tube and add 1 ml of
fresh lysozyme solution (12 mg/ml). Mix gently. Incubate in ice-water
bath for 20 min.
9) To each, add 12 ml of freshly prepared:
0.2 M NaOH
Mix thoroughly by inversion and incubate in ice-water bath for 10 min.
10) Add 7.5 ml of cold 3 M Na-acetate (pH 4.6). Mix carefully by
Incubate in ice-water bath for 20 min.
11) Centrifuge 30 min, 15K rpm, at 20¡C (room temp is OK).
12) Transfer supernatant to a 50 ml screw-cap tube (Corning, orange-cap).
Sometimes residual SDS can be seen to precipitate at this stage.
13) Add 100 ul RNase (10 mg/ml) and incubate at 37¡C for 20 min.
14) Extract 2 times with 12 ml phenol-chloroform (or until clean
then extract once with 12 ml chloroform-isoamyl alcohol (24:1). Gentle
vortexing to mix during the extractions is OK at this point because the
P1 should be mostly supercoiled and any E.coli genomic contamination
that will occur, already has. Centrifuge after each extraction 10 min
@ 3K at room temp.
15) After the last extraction, split each supernatant into two Oakridge
tubes and add 2 volumes of cold 100% EtOH to ppt. Leave overnight
at -20¡C. (There should be a total of 4 Oakridge tubes at this
16) Centrifuge 30 min, 15K rpm, at 4¡C. Pour off supernatant (save until
you check final yield). Use a marker to make a dot on the tube where
the pellet is.
17) Put 2 layers of parafilm over the tops of Oakridge tubes with the
pellet inside. Poke a few small holes in parafilm. Dry pellets in
a vacuum for @ 10 min.
18) Resuspend the pellets in TE or Water to a total volume of 1.5 or 2 ml.
I usually add about 0.5 ml to the first of the 4 Oakridge tubes and
pipet the pellet up and down until it's mostly dissolved. I then
transfer this solution to the second tube and repeat the process through
all four tubes. Store this in an eppendorf. It contains most of the
DNA. I then add a second 0.5 ml to the first tube, and repeat the
process through all 4 tubes to retreive any remaining DNA. A third round
with 0.5 ml gives a final volume of 1.5 ml that contains virtually all
* The DNA is usually yellowish or even dark yellow sometimes.
* There is often undissolved material after the final resuspension.
It doesn't seem to interfere in any subsequent reactions. It's probably
un-resuspended RNA and protein, which brings us to the next note...
* Even with the RNase step in this protocol, there is usually going to be
a LOT of e.coli RNA remaining mixed in with the P1 DNA. I wouldn't
a longer RNase step within the protocol. I just add a little to any
subsequent restriction digests using the P1 DNA. Alternatively, you can
add an RNase step and set of extractions after the final resuspension.
A final RNase step and set of extractions yields DNA clean enough to
give really good results on things like FISH (flourescence in-situ
hybridization) of metaphase chromosome spreads.
* For determining the approximate yield, I avoid spectrophotometry
because our spec is about 20 years old and doesn't like me). I prefer to
set up a a series of test-digests with a six-base restriction enzyme
(EcoRI or such). For example: 1 ul, 5 ul, 20 ul P1 DNA by EcoRI and
RNase, and maybe a couple of digests without RNase to see just how much
RNA actually is there. Run these out on a 1% agarose gel. It's also a
good idea to do this to verify the complexity of the P1 to make sure it
* Pellets can be frozen overnight at -20¡C after step 6 if desired, and the
protocol finished the next day.
Daniel.L.Burgess at um.cc.umich.edu
U of M Dept. of Human Genetics, Ann Arbor
Positional cloning...it's not the kill, it's the thrill of the chase.
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