Saturday, August 29, 2009

Rapid Boiling Method for Plasmid DNA Isolation

In the previous post, it had already explained about how to extract plasmid DNA by using alkaline lysis method. In this particular post, it will be explained the other method to extract plasmid DNA instead of using alkaline lysis method. It is Rapid Boiling method, an alternative to alkaline lysis method that was developed by Holmes and Quigley. Here, the cells are lysed partially allowing plasmids to escape, whereas the bacterial chromosomal DNA remains trapped in the cell debris. High temperature is then used to denature the chromosomal DNA, after which reannealing allows the plasmids to reassociate. Centrifugation removes the chromosomal DNA along with the cell debris, leaving the plasmid in suspension, from where it is recovered by isopropanol precipitation.

  • LB broth bacteria culture medium. The content are 1% Tryptone, 0.5% yeast extract, 200 mM NaCl, then it sterilize by autoclaving in suitable aliquots.
  • STET mix which is contain 5% (v/v) Triton X-I 00, 50 mM Tris-HCl, pH 8.0, 50 mM EDTA, pH 8.0, 8% (w/v) sucrose. Store the mix solution at room temperature.
  • Lysozyme: Dry powder. Store at -20oC.
  • 70% Ethanol.
  • Isopropanol.
  • TE solution: 10 mM Tris-HCl pH 8.0,1 mM EDTA.
  • A boiling water bath: An opened bottom tube rack is required because the tubes must be placed directly in the water to achieve rapid heating.
  • Sterile wooden toothpicks.

  • Set up a culture for each miniprep by inoculating 2-3 mL of L-broth, containing an appropriate antibiotic (e.g., 100 micrograms/mL ampicillin) with a bacterial colony. Grow overnight at 37oC with vigorous shaking.

    Where plasmids have a high copy number, the growth time may be reduced to approx 6 h.

  • Before starting the miniprep, begin boiling the water and make up a fresh solution of 1 mg/mL lysozyme in STET mix.
  • Fill a 1.5-mL labeled microfuge tube with an aliquot from each culture. Pellet the bacteria by centrifugation for 1 min at 12,000 g. Carefully aspirate off the supernatant using a drawn-out Pasteur pipet.

    The short centrifugation time leaves a loose pellet that is easier to resuspend. If the pellet does not readily resuspend, pipet the solution up and down to dislodge it. Do not suck the pellet directly into the pipet tip.

  • Vortex each pellet for a few seconds to break up the pellet. Add 20 micrliters STET mix to each tube. The pellet should now easily resuspend by vortexing.
  • Immediately place the tubes in the open-bottom rack, and place in the boiling water for exactly 45 s. Ensure that each tube is at least half submerged.
  • Centrifuge the tubes at 12,000 g for 10 min. A large, sticky, loose pellet should form.
  • Remove the pellet from each tube by "fishing" it out with a sterile wooden toothpick. Because 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.
  • Add 200 microliters isopropanol to each tube, and centrifuge at 12,000 g for 5 min.
  • Aspirate the supernatant, and wash the pellet in 500 microliters 70% ethanol. Centrifuge the tube for 1 min to compact the pellet, and then aspirate the 70% ethanol.
  • Air dry the pellets for 10 min, and resuspend each one in 100 microliters TE buffer. Vortex and shake for 10 min before use to ensure complete dissolution.
  • Use 10 microliters (equivalent to 100 ng of plasmid for most vectors) and analyze by gel electrophoresis.

It is possible to scale up procedures for the isolation of plasmid.
Now, you have two options method in extracting plasmid DNA, which are alkaline lysis method and Rapid Boiling method.

Reference: Number 18 on References