Small-Scale Preparation of Plasmid DNA (Miniprep)

(according to Sambrook-Fritsch-Maniatis: "Molecular Cloning" 1.25)

Harvesting Bacteria

  1. Transfer a single bacterial colony into 2 ml of ampLB medium (containing 50 µg/ml ampicillin) in a loosely capped 15-ml tube. Incubate the culture overnight at 37°C with vigorous shaking.
  2. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at 12,000 g for 30 seconds at 4°C in a microfuge. Store the remainder of the culture at 4°C.
  3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible. (The supernatant can be removed convenient with a diaposable pipette tip attached to a vacuum line

Hints

  1. Thaw competent cells on ice.
  2. Gently mix the cells by hand. Aliquot 100 µl of the cells into a prechilled 15 ml Falcon 2059 polypropylene tube.
  3. Add 1.7 µl (0.8 µl for JM109) of beta-mercaptoethanol provided with the kit or a fresh 1:10 dilution (stock 14.4 M) of beta-mercaptoethanol (diluted in high-quality water) to the 100 µl of bacteria, giving a final concentration of 25 mM.
  4. Swirl the contents of the tube gently. Incubate the cells on ice for 10 minutes, swirling gently every 2 minutes.
  5. Add 25 ng (0.1 - 50 ng (10 - 100 ng for TOPP cells)) of DNA to the cells and swirl gently. As a control add 1 µl of the pUC18 test insert to another 100 µl aliquot of the cells and swirl gently.
  6. Incubate the tubes on ice for 30 minutes.
  7. Heat pulse the tubes in a 42°C water bath for 45 seconds. The length of time of the heat pulse is critical for highest efficiencies.
  8. Incubate the tubes on ice for 2 minutes.
  9. Add 0.9 ml of preheated (42°C) SOC medium and incubate at 37°C for 1 hour with shaking at 225 - 250 rpm.
  10. When transforming the DNA, use a sterile spreader to plate 200 µl or less of the transformation mixture on the appropriate antibiotic plates. Incubate overnight.

Hints

Aliquoting Cells

When aliquoting, keep competent cells on ice at all times. It is essential that the Falcon 2059 tubes are placed on ice before the cells are thawed and that the cells are aliquoted directly into the prechilled tubes. It also important to use at least 100 µl of competent cells / transformation. Using a smaller volume will result in lower efficiencies.

Use of Falcon 2059 Tubes

It is important that Falcon 2059 tubes are used for the transformation protocol, since other tubes may be degraded by the beta-mercaptoethanol, which is ready to use. Using the beta-mercaptoethanol provided by the kit within three months is recommended. Use 1.7 µl (0.8 µl for JM 109) of the beta-mercaptoethanol provided or a fresh 1:10 dilution (sstock solution 14.4 M) / 100 µl of cells.

Quantity of DNA Added

Greatest efficiencies are observed when adding 1 µl of 0.1 ng/µl of DNA / 100 µl of cells. A greater number of colonies will be obtained when plating up to 50 ng, although the overall efficiency may be lower.

Length of Heat Pulse

There is a defined 'window' of highest efficiency resulting from the heat pulse in step 7 of the Transformation Protocol. Optimal efficiencies are observed when cells are heat pulsed for 45 - 50 seconds. Heat pulsing for at least 45 seconds is recommended to allow for slight variations in the length of incubation. Efficiencies decrease sharply when incubating for < 45 seconds or for > 60 seconds.

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