CHAPTER 32

The Rapid Boiling Method for Small-Scale Preparation

of Plasmid DNA

Adrian J. Harwood

1. Introduction The classic alternative to the alkaline lysis method for plasmid DNA

preparation (I; see Chapters 31 and 33) is that of Holmes and Quigley (‘21, and is commonly known as the rapid boiling method. This method is based on exactly the same principles as the alkaline lysis method. The cells are partially lysed, allowing plasmid molecules to escape, while most of the genomic DNA is trapped in the cell debris, which is then spun out. The remaining genomic DNA is removed by a denaturing step, in this case, high temperature rather than high pH, followed by rapid reannealing. As a consequence of their supercoiling, plasmid molecules are able to reassociate rapidly in these conditions, whereas genomic DNA remains denatured and is lost on the subsequent etha- nol-precipitation step.

This is a rapid and convenient method for making large numbers of small-scale plasmid preparations (minipreps). The DNA is of good qual- ity, and can be used directly for restriction enzyme digestion and subcloning. With only a small amount of further processing, it can be used as a template for DNA sequencing. The rapid boiling method, how- ever, is less convenient for large-scale preparations, for which the alka- line lysis method is better suited.

From* Methods m Molecular &o/ogy, Vol 58, Basic DNA and RNA Protocols Edited by A Harwood Humana Press Inc , Totowa, NJ

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266 Harwood

2. Materials 1. STET: 5% Triton X-100,50 mMTris-HCl, pH 8.0,50 mMEDTA, pH 8.0,

8% sucrose. Can be stored at room temperature. 2. Lysozyme: dry powder. Store at -20°C. 3. IsopropanoI:propan-2-01. 4. 70% Ethanol. 5. TE: 10 mM Tris-HCI, pH 7.5, 1 mA4EDTA. 6. A boiling water bath: A beaker of water heated by a bunsen burner will

suffice. An opened bottom tube rack is required because the tubes must be directly placed in the water to achieve rapid heating.

3. Methods 1. Set up a culture for each mnnprep by moculatmg 2-3 mL of L-broth,

containmg an appropriate antibiotic (e.g., 100 pg/mL ampicillin) with a bacterial colony. Grow overmght at 37°C with vigorous shaking (see Note 1).

2. Before starting the miniprep, start boiling the water and make up a fresh 1 mg/mL lysozyme STET mix.

3. Fill a 1.5-mL labeled microfuge tube with an aliquot from each culture. Pellet the bacteria by centrifugation for 1 mm at 12,000g (see Note 2). Carefully aspirate off the supematant on a water pump using a drawn-out Pasteur pipet.

4. Vortex each pellet for a few seconds to break up the pellet. Add 200 pL of STET to each tube. The pellet should now easily resuspend by vortexing (see Note 3).

5. Immediately place the tubes in the open-bottom rack, and place m the boil- ing water for exactly 45 s. Ensure each tube is at least half submerged.

6. Centrifuge the tubes at 12,000g for 10 min. A large, sticky, and loose pel- let should form.

7. Remove the pellet from each tube by “fishing” it out with a wooden tooth- ptck. Since the pellet is quite slippery, it is useful to have a paper tissue at the top of the tube to catch the pellet and prevent it from slipping back down into the tube.

8. Add 200 PL of isopropanol to each tube, and centnmge at 12,000g for 5 min. 9. Aspirate off the supematant, and wash the pellet in 500 PL of 70% ethanol.

Centrifuge the tube for 1 mm to compact the pellet, and then aspirate off the 70% ethanol

10. Air-dry the pellets for 10 min, and resuspend each in 100 pL of TE buffer. Vortex and shake for 10 min before use to ensure complete dissolution.

11. Use 2-10 pL (equivalent to 100 ng of plasmid for most vectors) per restriction enzyme digest and analyze by gel electrophoresis.

Rapid Boiling Method 267

4. Notes 1. If the plasmid has a high copy number, growth time can be reduced to

approx 6 h, enablmg the whole process to be carried out in a day. 2, The short centrifugation time leaves a loose pellet that is easier to resuspend. 3. If the pellet does not readily resuspend, pipet the solution up and down to

dislodge it. Do not suck the pellet directly mto the pipet tip.

References 1. Holmes, D. S and Quigley, M. (198 1) A rapid boiling method for the preparation

of bacterial plasmids. Anal. Bzochem. 114, 193-197.