Generating Minicircles

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Protocol

  • Grow an overnight culture of ZYCY10P3S2T containing the appropriate minicircle parental plasmid in 1-5 mL TB + Kan
    • Recipe for 50 mL Terrific Broth:
      • 1.2 g Yeast Extract
      • 1.0 g Tryptone
      • 200 uL glycerol
      • Up to 45 mL ddH2O
      • Autoclave at this point
      • Dissolve 11.6 mg of KH2PO4 and 62.7 mg of K2HPO4 into 5 mL of water and filter sterilize this solution directly into the autoclaved media
      • Alternatively, take 5 mL from a larger, pre-sterilized stock solution of potassium phosphates
  • In the morning, add an equal volume freshly made induction media to the culture
    • Recipe for Induction Media (for every 1 mL of overnight culture):
      • 1 mL LB
      • 40 uL 1M NaOH
      • 1-10 uL filter-sterilized 20% L-Arabinose solution
  • Incubate for 2-4 hours at 32oC with shaking
  • Isolate the minicircles using a Miniprep kit
    • NOTE: for optimal results, treat each 1 mL of culture as 5 mL of LB overnight culture, as these bacteria grow to a much higher density in Terrific Broth than do DH5a in LB. This can be performed as separate minipreps in separate tubes, or by following a modified Miraprep protocol
  • It is recommended to run a small sample out on a gel to ensure product is as expected. Minicircles can be cut with BamHI to produce a single band at 1.4-1.5 kB
    • If any other banding is seen on the gel (at larger MW), these plasmids can be degraded from the sample using the homing endonuclease I-SceI (stored at -80oC, works in CutSmart)
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