Molecular Cloning/Plasmid extraction

Growing Bacterial culture

Plasmids are almost always purified from liquid bacteria cultures, usually E. coli, which have been transformed and isolated. Virtually all plasmid vectors in common use encode one or more antibiotic resistance genes as a selectable marker (Ex :kanamycin, Ampicillin), which allows bacteria that have been successfully transformed to multiply uninhibited.

Harvesting and lysis of the bacteria

When bacteria are lysed under alkaline conditions both DNA and proteins are precipitated. Some scientists reduce the concentration of NaOH used to 0.1M in order to reduce the occurrence of ssDNA. After the addition of acetate-containing neutralization buffer the large and less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids can renature and stay in solution.

Preparations of plasmid DNA

Kits are available from varying manufacturers to purify plasmid DNA, which are named by size of bacterial culture and corresponding plasmid yield. In increasing order, these are the miniprep, midiprep, maxiprep, megaprep, and gigaprep. The plasmid DNA yield will vary depending on the plasmid copy number, type and size, the bacterial strain, the growth conditions, and the kit.

Minipreparation

Minipreparation of plasmid DNA is a rapid, small-scale isolation of plasmid DNA from bacteria. It is based on the alkaline lysis method invented by the researchers Birnboim and Doly in 1979. The extracted plasmid DNA resulting from performing a miniprep is itself often called a "miniprep". Minipreps are used in the process of molecular cloning to analyze bacterial clones. A typical plasmid DNA yield of a miniprep is 20 to 30 µg depending on the cell strain.

Miniprep Protocols

The protocol is described below.

Midipreparation

The starting E. coli culture volume is 15-25 ml of LB broth and the expected DNA yield is 100-350 µg.

Maxipreparation

The starting E. coli culture volume is 100-200 ml of LB broth and the expected DNA yield is 500-850 µg.

Small scale preparation of plasmid DNA or Miniprep

Inoculate a single colony of E. coli DH5-alpha cells bearing the plasmid in 5 ml of LB medium containing 100 µg/ml ampicillin (or other selection agent) and incubate overnight at 37°C with shaking at 200 rpm. Re-suspend the bacterial pellet from 3 ml of the overnight culture in 100 µl of ice-cold solution I (50 mM glucose, 25 mM Tris.HCl pH 8.0, 10 mM EDTA pH 8.0) by vortexing. Add 200 µl of freshly prepared solution II (0.2 N NaOH, 1% SDS) and the mix the contents by inverting the tube 4-6 times. Then 150 µl of ice-cold solution III (3 M potassium- 5 M acetate) was added and mixed by inverting the tube 4-6 times. Incubate the tube on ice for 5 min and centrifuged at 12,000g for 10 min at room temperature. The clear supernatant containing the plasmid DNA was collected into a fresh 1.5 ml eppendorf tube leaving the cellular debris behind and precipitated with two volumes of 95% ethanol for 5 min on ice. The precipitated plasmid DNA was pelleted by centrifugation at 12,000g for 20 min at room temperature, washed with 70% ethanol, air-dried and dissolved in 20 µl of water or TE pH 8.0 containing 20 µg/ml of DNase-free pancreatic RNase A (prepared by boiling RNase A for 20 min). The plasmid DNA was then checked on a 0.8% agarose gel and stored at -20C. The typical yield for a high copy plasmid from 3 ml of culture was about 12-16 μg and the DNA was suitable for routine procedures such as restriction enzyme digestion and preparation of radiolabelled probe.

Solution

Solution I

50 mM glucose,

25 mM Tris.HCl pH 8.0

10 mM EDTA pH 8.0

Solution II

0.2 N NaOH

1% SDS or SLS

Solution III

3 M potassium- 5 M acetate

RNase A

Ampicillin: A stock solution of 100 mg/ml ampicillin was made in sterile double-distilled water. Working concentration was 100 µg/ml. (concentration may vary lab to lab)

Kanamycin: A stock solution of 50 mg/ml ampicillin was made in sterile double-distilled water. Working concentration was 50 µg/ml.(concentration may vary lab to lab)