User:Norman Wang/Alkaline Lysis Plasmid Mini-Prep

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Materials

  • E. coli Containing Desired Plasmid(s)
  • Terrific Broth
  • Appropriate Antibiotics
  • Solution 1: GTE Solution
    • 50 mM glucose
    • 10 mM EDTA
    • 25 mM Tris-HCl (pH 8.0)
    • Autoclave mixture and store at 4°C
  • Solution 2: NaOH+SDS Solution
    • 0.2 M NaOH
    • 1% SDS
  • Solution 3: Potassium Acetate Solution
    • 50ml water
    • 29.5ml Acetic Acid, pH to 4.8 using KOH pellets
    • Water to 100ml
  • RNAse A
    • 10 mg/ml
  • 100% Ethanol
  • 100% Isopropanol

Procedure

  1. To a 125 mL culture flask, add 6 mL Terrific Broth, 6 uL antibiotics and a colony of bacteria. Include the tip, this helps with aeration. Shake ON at 37°C, 250 rpm.
  2. Add 1.5 mL of the culture to 3 eppendorf tubes.
  3. Centrifuge at 5,000 rpm for 3 minutes.
  4. decant supernatant.
  5. add 150 ul Solution 1, resuspend.
  6. add 300 ul Solution 2, invert 5 times
  7. add 225 ul Solution 3, invert 5 times—very slowly
  8. Centrifuge at 12, 000 rpm
  9. Combine the supernatant in 1 15ml tube. add 3 ul Rnase A, incubate at 55°C for 1 hour.
  10. Add ~1 mL Isopropanol, vortex for 3 seconds, leave in -20°C for 10 minutes
  11. Centrifuge at top speed for 15-30 minutes
  12. decant supernatant
  13. add 3 mL ethanol, with the tip, move the pellet around, incubate at room temperature for 10 minutes
  14. centrifuge top speed for 15-30 minutes
  15. decant supernatant, remove as much as possible without disturbing the pellet
  16. dry the pellet under vacuum. it is not necessary to use the laminar flow hood, you can use the solvent hood. Dry it at 37°C until the pellet is white.
  17. add 50 uL TE, place in 37°C to allow plasmids to dissolve. Add supernatant to a small tube for storage.

References

  • Margaret Ruzicka (personal communication) this above protocol gave really good yield on pSB1A3 containing ccdB insert.
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