Plasmid Miniprep

Posted on June 2nd, 2016 by UNM CC

M.Fero — 10/26/99

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)

  1. Add 20 µL of 4 M NaCl, mix. Add 100 µL of 13% PEG, mix. Incubate on ice x20 min.
  2. Spin at max speed, 4°C x15 min. Rinse pellet with 70% ethanol. Dry pellet.
  3. Resuspend in 50 µL H2O.

Tags: Fero-Bacteria

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