PEG?Preparation?of?Plasmid?DNA

PEG Preparation of Plasmid

Plasmid isolated by this procedure can be used routinely for electrophoretic analysis, restriction endonuclease digestion and transformation of E. Coli., sequencing, PCR and most other molecular biological techniques. The procedure is a modification of the rapid alkaline lysis method of Ish-Horowitz and Burke (1981).

You will need 3 basic solutions:-

Solution I: 50mM glucose, 25mM Tris.Cl, pH 8, 10mM EDTA

Solution II: 0.2M NaOH, 1%(w/v) SDS

Solution III: 3M potassium acetate, 2M acetic acid, pH4.8.

1) Grow overnight cultures of bacteria containing the plasmid of interest.

2) Pellet bacteria by centrifugation and resuspend in 0.02 culture volumes of Solution I.

3) Add to this suspension 0.04 culture volumes of Solution II. Mix contents of the tube by GENTLE inversion several times.

N.B.: Care must be taken to mix tube contents gently. This avoids mechanical shearing of bacterial genomic which could contaminate the resulting plasmid preparation.

4) Add 0.03 culture volumes of Solution III and invert 5-10 times to mix. Shake the tube vigorously for 5 seconds. Addition of Solution III causes renaturation of the genomic too rapidly and precipitation occurs.

5) Pellet bacterial debris by centrifugation at room temperature (maximum speed in a microfuge for 5 minutes for small volumes, 10,000g in a MSE Europa or equivalent for 20 minutes for large volumes).

6) Remove the clear supernatant to a clean tube.

7) Add DNase-free RNase A (0.01 supernatant volume of 10mg per ml stock), mix and incubate at 37°C for 30 minutes.

8) Phenol/chloroform extract to remove contaminating proteins and precipitate nucleic acids by the addition of an equal volume of propan-2-ol.

9) Pellet nucleic acids by centrifugation as described above (5).

10) Wash the once briefly in 70% ethanol and air dry for 5 minutes.

11) Re-dissolve in water or TE (10ul per ml of original bacterial culture), add an equal volume of 13% PEG 8000, 800mM NaCl, mix and incubate on ice for 30 minutes.

12) Pellet pure by centrifugation (10 minutes at room temperature in a microfuge or 20 minutes at 10,000g in a MSE Europa or equivalent), wash once in 70% ethanol and dry under vacuum.

13) Dissolve in appropriate volume of sterile, nanopure water or TE, pH 8.