Plasmid DNA Preparation and Purification using Celite Resin

P.R. Fisher, D.J. Fraser, M. Kotsifas & J.M. Santini.

School of Microbiology, La Trobe University, Bundoora, VIC 3083, AUSTRALIA.

Based on protocols from Merlin DNA Purification System by Ravi Iyer (riyer@rascal.bwh.harvard.edu) in bionet.molbio.methods&reagents 9311 #261.

and SIROglop SUPERnatural DNA minipreps by Robert Seymour & David Adelson, CSIRO Division of Animal Production, Prospect, NSW, Australia. (davead@prospect.anprod.csiro.au)

Introduction

Although plasmid purification kits abound, they are expensive - some more so than others of course. We now use this protocol to obtain high yields of clean plasmid DNA. It is very cheap, most of the cost being in the purchase of the Millipore spin filters for making the celite spin columns. Other brands/types of spin filter can be used, but the type specified here has provided us with the highest yields for the least cost.

Preparation of resin

To 67 g guanidine hydrochloride add 20 ml 3M potassium acetate pH4.8. Bring volume up to about 90 ml and adjust pH to 5.5 with NaOH (10M). Heat and stir to dissolve GuHCl while adjusting pH as necessary. Bring volume to 100 ml. Filter the hot GuHCl solution through Whatman filter paper (this step not necessary?) and add 10g Celite resin. Final GuHCl solution is ~7M. (Some GuHCl crystallizes out during filtration as the solution cools).

Preparation of Celite wash buffer

200 mM NaCl (in 200 ml, 2.34g)

20 mM Tris-HCl, pH 7.5 (in 200 ml, 20 ml of a 200 mM stock solution)

5 mM EDTA (in 200 ml, 4 ml of a 250 mM stock solution)

Dilute 1:1 with absolute ethanol.

Spin Columns

Use Millipore 5 µ m spin filters (UFC30SV00) - one filter per maxi-prep.

Alkaline lysis buffers

Resuspension buffer

50 mM Tris-HCl, pH 7.5

10 mM EDTA, pH 7.5

100 µ g/ml boiled RNAase A (boiled 30 min to inactivate DNAase).

Cell lysis solution

0.2 N NaOH

1% SDS

(Prepare fresh from 10x stock solutions).

Neutralizing buffer

3M Potassium acetate, pH 4.8.

Maxi-prep protocol

Grow (shaken at 37C overnight) 300-500 ml of E. coli bearing the plasmid in LB or nutrient broth supplemented with the appropriate antibiotic.

Harvest the bacteria by centrifugation (eg. 3000 rpm/15 min in swing out rotor of bench top Sorvall centrifuge).

Thoroughly resuspend the cells in 10 ml Resuspension Buffer, transferring them to a 50 ml Falcon tube.

Add 10 ml Cell lysis solution and mix by inverting several times.

Add 10 ml Neutralizing buffer and mix thoroughly.

Spin to pellet precipitate (eg. 3000 rpm/30 min in swing out rotor of bench top Sorvall) and transfer supernatant to two 50 ml centrifuge tubes for the Sorvall SS34 rotor (~15 ml per tube).

Add an equal volume of isopropanol and mix by inversion to precipitate the DNA, optionally placing the tubes at -20C for 30 min, before spinning in the Sorvall SS34 rotor at 10,000 g for 30 min to pellet the DNA. Discard the supernatant.

Dry the pellet by placing the inverted centrifuge tube on adsorbent paper at room temperature for 5-10 min.

Take up and pool the DNA from both tubes using 500 µ l of Celite resin (use a blue tip with a few mm of the tip snipped off with scissors) and transfer it to a Millipore spin column.

Spin for 2 secs in the Eppendorf centrifuge, discard the liquid in the bottom tube and wash the resin 3 times by spinning through 400 µ l Celite Wash Buffer.

Elute the DNA from each spin column with 400 µ l TE (optionally prewarmed to 60C) and retain the eluate.

If desired, precipitate the DNA by adding 40 µ l sodium acetate (3M, pH 5.5) and 1.0 ml of absolute ethanol, then washing with 70% ethanol, drying in the Speed-E-Vac (5 min) and dissolving the DNA in the desired volume of TE.

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