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
- Recipe for 50 mL Terrific Broth:
- 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
- Recipe for Induction Media (for every 1 mL of overnight culture):
- 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)