Cesium chloride plasmid isolation
1.
Set-up a 3 ml LBAmp (cf=100 µg/ml) overnight of each sample. Incubate at 37C with
shaking.
2. Inoculate 100 ml of TB (terrific broth) medium containing antibiotics with a 1:1000
dilution of the overnight from step 1. Incubate at 37˚C for 24 hours with vigorous shaking
in a 500 ml flask.
3. Pellet in 250 ml Sorvall bottles at 7000 rpm for 10 min at 4˚C
4. Decant the medium
5. Resuspend each pellet in 20 ml HS solution and transfer to an Oakridge tube and pellet at
7000 rpm for 10 min. at 4˚C
6. Decant the supernatant
7. Resuspend the pellet by vortexing in 8 ml of Solution I + 800 µl of 50 mg/ mL lysozyme at
room temp.
8. Incubate for 10 min at room temp
9. Add 16 ml of freshly prepared Solution II. Close the tube and mix the contents by inverting
the tube rapidly several times (DO NOT VORTEX).
10. Incubate for 10 min on ice.
11. Add 12 ml of ice cold Solution III. Close the tube and mix the contents by inverting the
tube 50-60 times
12. Incubate for 10 min on ice
13. Centrifuge for 20 min at 15K at 4˚C (after spin set temp. to 25˚C)
14. Transfer the supernate in equal portions to 2 new Oakridge tubes being careful to avoid any
of the white flocculent material. (If surface of supernate has pellicle, use sterile wooden
stick to remove any floating material)
15. Add 0.6 volumes (~12 ml/tube) of room temp isopropanol. Mix by vortexing. Let sit at
room temperature for 20 minutes.
16. Centrifuge for 30 min at 15K at room temperature.
17. Decant the supernatant carefully to avoid losing the pellet (decant into separate beaker for
each plasmid so DNA can be retrieved if needed) and allow the pellet to drain 5 mins.
CAN STOP AT THIS POINT (store DNA pellet at –20˚C)
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Redissolve each pellet completely in 5 ml of TE (pH 8.0) and combine into one tube.
Add 16.8 ml of TE (pH 8.0) to each sample.
Add 26.88 gm of CsCl to each tube and dissolve. (The reaction is endothermic.)
Add 2.67 ml of EtBr (10.0 mg/ml in water) to each sample. Keep the samples in subdued
light from this point on. (The final density should be 1.55 gm/ml)
Mix the contents thoroughly by inverting the tube several times.
Spin the tubes at 18K for 30 min in Oak Ridge tubes. to pellet flocculent material which
forms upon the addition of EtBr.
Transfer the contents to a 36 ml Beckman polyallomer with a 10 ml pipette.
Balance the tubes to within 0.01 g and seal them (use fake tube containing TE, CsCl and
water in place of DNA and EtBr).
Transfer the tubes to a VTi-50 rotor, spin at 45K for 18-22 hours at 15˚C (acceleration speed
for obtaining speed=N; acceleration speed for stopping=1).
After centrifugation the following bands should be easily discernible: (from the bottom up)
a) closed circular plasmid DNA
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b) nicked plasmid DNA / linear DNA
The closed circular DNA is collected from the centrifuge tube by puncturing the tube with a
10cc syringe (fitted with an 18 gage needle), just below the plasmid DNA band (remember
to unseal the centrifuge tube) and withdrawing the band slowly into the syringe. For better
visualization of the various bands the collection may be carried out under longwave UV
light.
Ethidium bromide (EtBr) is removed from the DNA by extracting 6 times with an equal
volume of H2O saturated n-butyl alcohol (or until pink color is gone). This is done in the
same syringe that was used to remove the plasmid band from the centrifuge tube.
The plasmid samples are then dialyzed vs. four changes of TE buffer, pH 8.0 [3X 1
hour/change then 1 change overnight at 4°C] and then precipitated by addition of 0.1 vol of
3 M NaOAc and 2.0 vol of EtOH. Incubate on ice for 30 min or overnight at –20˚C.
The precipitate is collected by centrifugation at 15K rpm at 30˚C for 30 min. The pellet is
redissolved in 1 ml of TE, pH 8.0, and quantitated by A260 measurement and analyzed by
agarose gel electrophoresis
Solutions:
TB (Terrific Broth):
Solution I:
For 900 ml
12 g bacto-tryptone
24 g bacto-yeast extract
4 ml glycerol
QS to 900 ml
Solution II:
For 100 ml
2.31 g KH2PO4 (Cf = 0.17 M)
12.54 g K2HPO4 (Cf = 0.72 M)
QS to 100 ml
Solution I:
50 mM glucose
10 mM EDTA
25 mM Tris-HCl (pH 8.0)
5 mg/ml lysozyme (added just prior to use)
Solution II:
0.2 N NaOH
1% SDS
Solution III:
(the solution is 3M with respect to potassium and 5M with respect to acetate)
60 ml 5M Potassium acetate
11.5 ml glacial acetic acid
28.5 ml diH2O
TE buffer:
10 mM Tris-HCl (pH 8.0)
1 mM EDTA
HS buffer:
10 mM Hepes (pH 7.3)
.85% NaCl