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2021-02-01T19:44:17Z

Created page with "= 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 Ye..."

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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)

Generating Minicircles - Revision history

Admin: Created page with "= 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 Ye..."

Admin: Created page with "= 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 Ye..."