A27 Small Scale Plasmid Isolation; "Miniprep"

To isolate plasmid DNA on a small-scale.

Prior Work

Transformed bacteria need to be available (A-22).

Materials

• Phenol
  • Note the many cautions given for handling phenol. Purchase high quality phenol. Melt the phenol at 68°C. Add 100 mg
  • 8-hydroxyquinoline for every 100 g phenol. The yellow substance imparts color, inhibits RNase, and chelates metal ions. Extract the melted phenol several times with an equal volume of 1.0 M Tris (pH = 8.0). Follow with extractions using 0.1 M Tris containing 0.2% β-mercaptoethanol until the pH of the aqueous phase remains above pH 7.6. Store the phenol under equilibration buffer at 4°C.
  • Some protocols suggest that water-washed phenol is more stable than buffer-washed phenol. These protocols call for washing the phenol with water and storing at 4°C.
• Chloroform:Isoamyl alcohol
  • 24 volumes reagent grade chloroform
  • 1 volume reagent grade isoamyl alcohol
    • This mixture is stable for long periods of time at room temperature.
• Solution A
  • 50 mM glucose
  • 10 mM EDTA
  • 25 mM Tris-HCl (pH 8.0)
• Solution B (must be made fresh)
  • 0.2 M NaOH
  • 1% SDS
• Solution C
  • 60 mL 5 M KOAc
  • 11.5 mL HOAc (glacial)
  • 28.5 mL water

Background

Suppose some bacteria that have been transformed using a plasmid vector containing recombinant DNA are growing. It is important to confirm that the plasmids being produced contain the desired DNA. For example, during the transformation, a polydisperse source of DNA may have been used so that it is not assured that any of the growing bacteria contain DNA of interest. A miniprep is a useful preliminary step to characterizing the plasmid DNA. It might be performed after a colony lift in anticipation of an analysis and, subsequently, selecting particular bacterial colonies to grow. Several prepared reagents are required for minipreps.

Procedure

IMPORTANT

Phenol is extremely toxic. Wear gloves, goggles, and aprons when using phenol. Phenol causes burns. Phenol is absorbed through the skin. If skin comes in contact with phenol, wash immediately with large amounts of water. Scrub affected area with soap and water. DO NOT use a solvent such as alcohol; this dissolves the phenol and increases the amount that will penetrate the skin. Seek medical attention after the washing process.

1. Pipetting will be performed throughout the plasmid isolation procedure. Refer to instructions for pipetting (A-02). All pipet tips are autoclaved before use. Autoclaving kills microorganisms and inactivates nuclease enzymes. Discard used pipet tips and replace with fresh tips before each pipetting.
2. Depending upon the nature of the particular experiment, there is a potential biohazard. Both the bacteria and the plasmids present a potential hazard. For this reason, gloves may be required throughout the procedure.
3. Gloves are required to protect workers from chemical hazard during the phenol extraction procedure. Gloves are required to protect the plasmid from environmental contamination during some procedures.
4. The acetate buffer system and "solution A" are cooled. Cooling suitable amounts in conical microcentrifuge tubes speeds the cooling process. Do not cool the SDS solutions; they may precipitate. Bacteria are grown overnight. See A-09 for experimental suggestions.
5. Pipet aliquots of bacterial cultures into conical microcentrifuge tubes. Dispose of pipet tips in a waste can labelled for biohazard. Return the unused bacterial culture to ice. Cap the tubes. Microcentrifuge for 15 seconds at room temperature. Note that a pellet of bacteria forms. The pellet will be resuspended, and the plasmids isolated from the bacteria.
6. Use a pipet to aspirate the supernatant fluid. Return the supernatant fluid to the original culture tube. Do not discard this fluid at the sink. Add 100 mL of "Solution A" to each pellet. Cap the tubes. Touch the capped conical microcentrifuge tube to a vortex mixer until the pellet is resuspended. The time required varies depending upon several factors, especially the previous centrifugation time. No pellet is visible after sufficient vortexing. Wait for 5 minutes.
7. Prepare the lysing solution using equal amounts of 0.4 M NaOH and 2% SDS (sodium dodecyl sulfate). Treatment with the lysing solution lyses the cells and denatures all of the DNA present. Add an aliquot of the lysing solution (usually 200 µL) to each tube. Cap each tube and mix. Note the mixing technique carefully. Do not use a vortex mixer, as that might damage the denatured plasmid DNA. Store the mixed material on ice for 5 minutes. The preceding sequence is very important. Review the technique again if necessary.
8. Set the timer, usually for 5 minutes.
9. Acidification with 150 µL of cold acetate buffer leads to precipitation of chromosomal DNA but allows the plasmid DNA to renature. Cap and mix gently. Note the formation of precipitate upon mixing.
10. Store on ice; set the timer, usually for 5 minutes. Microcentrifuge for 5 minutes at 4°C. Use a pipet to transfer the supernatant fluid containing the renatured plasmid DNA to a fresh microcentrifuge tube. Denatured chromosomal DNA is left behind. Discard both the pipet tip and the microcentrifuge tube containing the precipitate with materials to be autoclaved.
11. Perform the following procedure in a hood. Be certain to wear both eye protection and gloves. Wear an apron in addition to a laboratory coat. Phenol extraction removes much of the protein material. Keep the prepared phenol in the refrigerator or on ice until just before use. Place the tip of the pipet through the water layer and below the surface of the phenol layer. Remove an aliquot of the same volume as the total volume of liquid to be treated. Dispose of the pipet tip used to dispense phenol in a container dedicated to phenol disposal. Add an aliquot of chloroform/isoamyl alcohol solution to the mixture in each tube. The volume of the aliquot should be the same as that of the phenol added.
12. (Some protocols call for mixing the phenol with the chloroform, and using the resulting solution instead of using phenol followed by chloroform.)

    REMEMBER THAT PHENOL ALWAYS REQUIRES ATTENTION TO TECHNIQUE; IT IS A PARTICULARLY DANGEROUS SUBSTANCE.

13. The tube containing the phenol must be shaken; special caution is necessary. Mix each tube using a vortex mixer. Renatured plasmid DNA can survive vortex mixing. After vortex mixing, only one layer is seen in the tube. The tubes containing phenol are now removed from the hood and brought to the microcentrifuge. Remember to maintain eye protection and gloves. Microcentrifuge for 2 minutes at room temperature.
14. Remove the tubes from the microcentrifuge. Note that two layers have formed. The top layer contains the plasmid DNA. Place the microcentrifuge tubes in a tray, and return to the hood. Use a pipet to transfer the aqueous layer -- the top layer -- to a fresh microcentrifuge tube.
15. Discard the tube containing the phenol and the used pipet tip into a container marked "Phenol Disposal". (Do not discard phenol in ordinary trash. The material is too toxic, and risks to janitorial personnel are too great. Make arrangements with commercial waste disposal providers for the disposal of phenol, if possible. A large-scale disposal procedure is to dissolve the phenol in a flammable solvent, and burn the resulting mixture in a furnace equipped with an afterburner. Consult the safety officer at your laboratory if phenol disposal is handled internally.)
16. Add an aliquot of 100% ethanol (room temperature) to the aqueous layer. The volume of the ethanol should be twice that of the aqueous layer. Cap the tube. Mix using a vortex mixer. Set the timer for 2 minutes. Wait. Microcentrifuge for 5 minutes at room temperature. The precipitate contains RNA and plasmid DNA. Uncap. Drain the liquid into a beaker for disposal and press the tube on a paper towel to remove as much liquid as possible.
17. Add 1 mL of 70% aqueous ethanol at room temperature. Cap and vortex mix briefly. Microcentrifuge for 5 minutes at room temperature. Uncap. Drain the liquid into a beaker and shake any excess liquid onto a towel as earlier. Set upside-down on the towel for drainage.
18. Cover each drained tube with Parafilm®. Use a syringe needle to poke tiny holes in the Parafilm®. Place the covered tubes in a vacuum desiccator containing anhydrous calcium chloride. Apply a vacuum through a potassium hydroxide drying column for about 10 minutes. A lyophilizer may be used if available. Use caution with glassware under evacuation. The device shown is made of plastic. Tape or otherwise guard glass devices. Remove the vacuum. Open the system, and remove the tubes. Remove the Parafilm®. Take up the pellet in sterile distilled water or 1x TE buffer. The volume of solvent varies depending upon the size of the pellet. Use from 20 to 50 µL.

Going Further

At this point, the small scale plasmid isolation is complete. A restriction analysis (A-28) follows.

References