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 32^oC 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 -80^oC, works in CutSmart)

Generating Minicircles