CsCl Gradient for large Scale Preparation of Plasmid DNA
Inoculate 5 ml LB + appropriate antibiotic with a single colony of E. coli strain harboring the plasmid to be isolated. Grow at 37oC for 6-8 hours with vigorous shaking.
Inoculate 25 ml LB + appropriate antibiotic with 1 ml of culture from previous step. Grow at 37oC overnight with vigorous shaking.
In the morning inoculate 500 ml LB + appropriate antibiotic with 10 ml of the overnight culture and grow for at least 18 hrs at 37oC with vigorous shaking.
Harvest cells by centrifugation 5000-6000 RPM in a JW 8.1000 for 10 min at 4oC.
Decant supernatant and briefly wash pellet with cold water to remove media.
Resusupend pellet in 5 ml Solution I and transfer to a 30 ml SS34 centrifuge tube, add 25 mg lysozyme, stir solution with a Pasteur pipette. Let solution stand at room temperature for 10 minutes.
Add 10 ml freshly prepared Solution II. Stir solution extensively until the solution becomes homogenous. Let solution stand on ice for 10 minutes.
Add 7.5 ml Solution III. Stir solution extensively. A white fluffy precipitate will form. Let stand on ice for 10 min.
Centrifuge 10 min at 13,000 rpm (Sorvall SS34 Rotor) at 4oC.
Decant supernatant through cheese cloth or gauze into a clean centrifuge tube.
Add 0.6 vol isopropanol, mix by inversion, and allow solution to stand at room temperature for 10 min.
Centrifuge for 10 min at 11500 rpm (Sorvall SS34 Rotor) at 4oC.
Decant supernatant and wash pellet with 70% EtOH.
Centrifuge for 10 min at 11500 rpm (Sorvall SS34 Rotor) at 4oC.
Decant Supernatant and allow pellet to dry in the centrifuge tube inverted.
Resuspend pellet in 4 ml TE buffer and transfer to 15 ml Falcon Tube.
Add Exactly 4.4 g CsCl to the solution. Mix by inversion making certain that all the CsCl is dissolved.
Add 400 ml 10 mg/ml EtBr and mix thoroughly. Note: EtBr is a mutagen use extreme care when handling this solution.
Add solution to an optiseal tube (Beckman part number 362185). A balance tube must be created that contains the identical CsCl/EtBr concentrations. This balance should be within 0.05 g of the CsCl gradient tube. Note: Severe damage and personal injury can result if the balance is not within 0.05 g or the balance does not contain a CsCl/EtBr gradient.
Cap tubes and place in Beckman rotor VTi-90 add spacer (Beckman part number 362198). Fix and screw gasket into the rotor using a torque wrench to exactly 120 in-lbs of torque. Note: we have other spacers in the lab it is essential to use spacer 362198, this spacer is gold in color. Other spacers will leak at high speeds.
Place rotor in ultra centrifuge set to 65,000 rpm. Centrifuge for at least 18 hrs at 20oC.
Disassemble rotor and remove tubes. Clamp tube, near the top of the tube, to a ring stand.
Place a needle in the top of the tube to allow air to enter.
Turn on UV light (Long wave) and extract the bottom (plasmid) band from the tube.
Place Extracted plasmid into a 15 ml conical tube and add an equal volume of NaCl Saturated/n-butanol. Vortex for 10 seconds and allow tube to settle for 2 min. Pipette off top layer and dispose of in appropriate waste container (EtBr Waste under the fume hood). Repeat this step until aqueous phase is clear.
Bring volume to 8 ml using TE buffer. Transfer to a centrifuge tube and add 16 ml EtOH and place at -20oC for at least 30 min.
Centrifuge for 30 min at 11500 rpm (Sorvall SS34 Rotor) at 4oC.
Decant supernatant and wash pellet with 70% EtOH.
Centrifuge for 10 min at 11500 rpm (Sorvall SS34 Rotor) at 4oC.
Decant supernatant and allow pellet to dry.
Resuspend pellet in 250-500 ml TE Buffer.
Measure concentration, A260/A280 ratio and run an agarose gel to analyze purity.
Solution I
50 mM Glucose 9 g Glucose
25 mM Tris pH 8.0 25 ml 1 M Tris pH 8.0
10 mM EDTA pH 8.0 20 ml 0.5 M EDTA pH 8.0
ddH2O to 1 L
Solution II
0.2 N NaOH 0.4 ml 10 M NaOH
1% SDS 1 ml 20% SDS
ddH2O to 20 ml
Solution III
3 M Potassium Acetate 294 g Potassium Acetate
1.18 M Formic Acid 54.3 g Formic Acid
ddH2O to 1 L
Robertson Lab