Plasmid Miniprep

 

Materials

Solution II: 0.2N NaOH/1% SDS

Solution III: 3 M KOAc, pH4.8

RNAseA (DNAse free) 10 µg/mL

Chloroform/Isoamyl alcohol (1/25 v/v)

Isopropanol

70 % ethanol

13% PEG8000

Procedure

1) Culture 5 mL of bacteria o.n. in LB + 100 mg/mL ampicillin.

2) Spin down 1.5 mL of each culture (5,000 rpm x 3 min.) Discard supernatant. Add additional 1.5 mL of cell suspension and spin a second time.

3) Resuspend pellet in 100 µL of H2O.

4) Add 300 µL of Solution II. Mix, incubate on ice for 5 min.

5) Add 300 µL of Solution III. Mix, incubate on ice for 5 min.

6) Spin 5 min at maximum speed, 4°C. Save supernatant in a fresh tube.

7) Add 10 µL of RNAseA, incubate at 37°C x5 min.

8) Extract with 1 volume of chloroform x1, spin 2 min. Transfer aqueous phase to a new tube.

9) Add 1 volume of isopropanol, mix. Spin at max. speed x 2 min.

10) Rinse with 70% ethanol. Dry pellet. Resuspend in 80 µL of T.E.

 

Optional (for DNA sequencing)

11) Add 20 µL of 4 M NaCl, mix. Add 100 µL of 13% PEG, mix. Incubate on ice x20 min.

12) Spin at max speed, 4°C x15 min. Rinse pellet with 70% ethanol. Dry pellet.

13) Resuspend in 50 µL H2O.