(modified from Sigi Ruppert version 1.0)
Day One:
Inoculate a 2 L flask
containing 500 ml LB +Amp (500 ul Amp 100 mg.ml) with a single colony. Grow at 37 C overnight on shaker.
Day Two:
1.
Spin down bacteria
in 500 ml centrifuge bottles at 6000 rpm in JA-10 rotor, 5 min.
2.
Resuspend pellet in
10 ml Solution 1 using a 10 ml pipet.
3.
Add 20 ml Solution
2 and swirl to mix. Incubate at RT
5 min.
4.
Add 15 ml cold
Solution 3, swirl well and incubate on ice 15 min.
5.
Spin in 500 ml
centrifuge bottles at 6000 rpm in JA-10 rotor, 10 min.
6.
Using a funnel, filter
the sup through 2 layers of Kimwipes into a clean 5o ml centrifuge tube.
7.
Add 27 ml
isopropanol (0.6 vol), and leave at RT 10 min.
8.
Spin in JA-10 at
6000 rpm, 10 min.
9.
Pour off isopropanol
then add 5 ml 80% EtOH.
10.
Spin 6000 rpm, 5
min.
11.
Pour off ethanol
and dry pellet inverted for about 5 min then wipe excess alcohol off sides of
tube.
12.
Dissolve the pellet
in 4.3 ml 1X TE and transfer exactly 4.3 ml of the DNA/TE solution to a 15 ml conical tube.
13.
Add exactly 4.84 g CsCl. If you have only one DNA sample, make a balance tube with
4.2 ml TE and 4.5 g CsCl.
14.
Invert gently several
times to resuspend CsCl.
15.
Add 0.2 ml ethidium
bromide (10 mg/ml in water).
16.
Spin 5 minutes at
full-speed in the table top centrifuge.
17.
Transfer the cleared
liquid to a Beckman quick seal tube.
Use a pair of short Pasteur pipettes and fill only to the base of the
neck.
18.
Weigh the tubes and
make sure the corresponding tubes are within 0.01 g of each other.
19.
Seal the tops with
the heat sealer‹check for leaks after sealing by squeezing.
For one day prep:
Spin
in Vti80 at 80,000 rpm for 4 hours at 20C.
For overnight prep:
Spin
in either Vti80 or Vti65 at 55,000 rpm for 12 to 16 hours at 20C.
After the spin is
complete:
20. Collect the lower
band (containing the closesd circular plasmid DNA) using an 18G needle and a syringe. First, vent the top of the tube with a 25G needle, then
use the 18G needle and a syringe to gently draw out the lower band.
Gently squirt the DNA into a 15 ml conical tube. Discard the rest of the ultracentrifuge tube into
the CsCl/ethidium bromide waste.
21. Extract the
ethidium bromide from the DNA with water saturated 1- butanol. Add an equal volume of 1-butanol, shake
and allow to separate. Remove and discard the TOP phase that
contains the ethidium and
2-propanol. Extract until the top
phase is no longer pink, then extract 2
more times.
22.
Dilute the DNA
containing solution with 4 volumes of 1X TE.
23.
Add 2.5 volumes
EtOH and put at 20C for atleast 30 minutes.
24.
Spin in JA-10 rotor
at 9000 rpm for 20 minutes.
25.
Remove sup then
wash pellet with 3 ml 80% EtOH, spin 5 minutes at 9000 rpm.
26.
Remove sup and dry
pellet.
27.
Resuspend in 250
500 ul 1X TE.
Solution 1: 50
mM glucose (also called dextrose)
25
mM Tris pH 8
10
mM EDTA pH8
per
liter: 9.91 g glucose, 25 ml
1M Tris pH 8, 20 ml 0.5M EDTA, water
to 1 L.
Solution 2: 0.2
M NaOH
prepare fresh 1%
SDS
per
100 ml: 88 ml water, 10 ml 10%
SDS, 2 ml 10 M NaOH
Solution 3: 2.5
M potassium acetate
2M
acetic acid
per
500 ml: 147 g potassium
acetate, 60.05 ml glacial acetic acid, water
to 500 ml.
Water-saturated
butanol:
Add
30 ml sterile water to 220 1-butanol and shake vigorously. Allow to sit overnight
to allow the water to saturate and separate from the butanol.