An inexpensive and simple method for DNA purifications on silica particles

We have developed a simple, rapid and cheap method for the isolation of DNA from various sources by a further development of the silica binding method of Boom et al. (1). In combination with the alkaline lysis method for preparation of bacterial lysates, this constitutes a rapid and effective method for the isolation of plasmid DNA for sequencing and other purooses. In this method the DNA-binding matrix is provided by diatomaceous earth as a cheap and convenient source of silica particles. Pretreatment with nitric acid is not required, but the diatoms should be size fractionated. Very small particles are difficult to remove without filtration and may inhibit some enzymic reactions. This is performed by resuspending 50 g of diatoms (Sigma D 5-384) in 500 ml of water, and allowing them to settle in a 500 ml measuring cylinder for 3 hours. The settled particles are recovered in 10—20 ml and resuspended at 10 mg/ml in 4 M guanidine thiocyanate (Sigma G-6639), 50 mM Tris-HCl pH 7.0, 20 mM EDTA (assuming no significant loss of material during the size fractionation step). This solution (termed DNAbind) should be stored in the dark and is stable for at least three months. DNA may be recovered from solution (eg enzyme reaction mixtures or gel eluates) by the following method which is designed for a volume of DNA solution of 0.5 ml. If the sample volume is smaller than this it should be adjusted to 0.5 ml with water and transferred to a 1.5 ml microfuge tube. The DNAbind solution is shaken and 1 ml added to the sample. The tube is mixed for 2 min at room temperature to allow binding of the DNA before the diatoms are sedimented for 5 —10s in a microfuge. The supernatant is removed with a drawn-out Pasteur pipette and the pellet resuspended in 50% ethanol containing 200 mM NaCl, 10 mM EDTA, 50 mM Tris-HCl pH 7.4. Diatoms are pelleted again, and the washing step repeated. The final pellet is washed in 1.0 ml of acetone as above and briefly dried at 65°C. DNA is eluted by resuspending the pellet in 50-200 /d of water and incubating at 65 °C for 2 minutes. Diatoms are then removed by sedimentation and the aqueous solution of DNA transferred to a fresh tube. We have successfully recovered ds DNA fragments as small as 120 bp. This procedure is compatible with a modification of the alkaline lysis method for plasmid minipreparation and considerably speeds this process (2). We routinely resuspend 1.5 ml of an overnight bacterial culture in 200 /il of 25 mM Tris-HCl pH 7.4, 10 mM EDTA. If required RNAse A can be included in this solution at 50 /tg/ml. Cells are lysed by the addition of 200 /tl of 0.2 M NaOH, 1 % SDS and gentle inversion. When the suspension has cleared 0.2 ml of 2.55 M potassium acetate pH 4.8 is added and mixed. The flocculent precipitate is removed by centrifugation and the supernatant transferred to a clean tube. 1 ml of DNAbind solution is added and processed as above. This method yields in excess of 5 fig of DNA from a typical minipreparation of plasmid pTZ18R and requires 10-15 minutes to perform. Total costs involved (plasticware and chemicals) are low (approximately 12p per minipreparation). Single-stranded DNA is also recoverable from PEG-precipitates of bacteriophage after resuspension of the pellet in 0.5 ml of water. We have found DNA prepared this way to be immediately suitable for most laboratory procedures: both single and doublestranded sequencing reactions, restriction enzyme digestion, ligation, and for transfection of eukaryotic cells using lipofectin (data submitted but not shown). The method is suitable for the recovery of DNA between enzyme reactions and we estimate the efficiency as 60—80%. However losses become appreciable after 4 successive re-isolations. DNA made this way is lost during phenol extraction unless acidified beforehand by addition of sodium acetate pH 5.0 to 0.1 M. No further salt addition is then required for ethanol precipitation.