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PlasmidMiniprep
PlasmidMiniprep

Plasmid Minipreps

This protocol is used for preparing small amounts of plasmid (a few μg) suitable for basic analysis like restriction digestion from small cultures of E. coli. It is fast, inexpensive and reliable.

  1. innoculate a 3 ml culture of media with appropriate antibiotics and grow overnight with shaking.
    • rich media like Circlegrow will give higher yields than LB. yield will also depend on the size of the plasmid and the nature of the plasmid origin
  2. transfer 1 ml of the grown culture to an eppendorf tube, store the remaining culture at 4C.
    • the culture at 4C will remain viable for at least one week
  3. microfuge the 1 ml culture at 12000g for one minute and aspirate off the supernatant.
    • the pellets can be frozen at -20C indefinitely
  4. resuspend the bacterial pellet in 100 μl of Solution I by pipetting up and down.
    • the bacteria can be left suspended in Solution I at room temperature for up to an hour
  5. add 200 μl of Solution II and mix by flicking and inverting the tube. place on ice for from 2 to no longer than 5 minutes until the solution clears.
    • this step lyses the bacterial membrane and cell wall and denatures both plasmid and genomic DNA. if left too long at this stage, the plasmid DNA will denature irreversibly, making it useless for subsequent analysis
  6. add 150 μl of Solution III and vortex vigorously. place on ice for 5 minutes or longer.
    • the potassium salt of SDS is relatively insoluble and forms the bulk of the precipitate. the bacterial genomic DNA is large enough to be trapped in this precipitate and removed. the much smaller plasmid DNA is not trapped and renatures after the acetic acid in solution III neutralizes the hydroxide from Solution II
  7. microfuge at 12000g for three minutes.
  8. transfer 400 μl of the supernatant to a fresh eppendorf tube, taking care to avoid any precipitate.
  9. add 800 μl 100% ethanol, vortex, sit at room temperature 2 minutes, then microfuge at 12000g for 3 minutes
  10. discard the supernatant, rinse the pellet with 500 μl 75% ethanol, discard the rinse
  11. spin down and aspirate dry, resuspend in a suitable volume of aqueous solution (100 μl - 200 μl)


Solutions

Solution I
50 mM glucose
25 mM Tris pH 8.0
10 mM EDTA pH 8.0
prepare in batches of 100 ml, store at room temperature

Solution II
0.2N NaOH freshly diluted from a 10 N NaOH stock stored in a plastic (polypropylene) bottle
1% SDS

Solution III
60 ml of 5 M potassium acetate
11.5 ml of glacial acetic acid
28.5 ml H2O
prepare in batches of 100 ml, store at room temperature
The resulting solution is 3M with respect to potassium and 5M with respect to acetate


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