MC-Easy Minicircle DNA Production Kit (does not include ZYCY10P3S2T producer E. coli strain)

The quick and easy MC-Easy minicircle preparation protocol

Going from parental minicircle plasmid ready-for-maxi-prep pellet is a simple, 2-day process with the MC-Easy Kit:

Day 1

1. Inoculate 2 mL of LB/Kan with ZYCY10P3S2T Minicircle Production Cells that have already been transformed with the parental minicircle plasmid, and grow for 1 hour at 30°C.
2. Use the 2 mL culture to inoculate 200 mL of LB/Kan and shake for 16 hours (or overnight) at 30°C.

Day 2

3. Add 200 mL Induction Medium (included in the MC-Easy Kit), and shake for 5.5 hours at 30°C.
4. Transfer culture to centrifuge bottle and spin at 1,500g for 15 minutes. Decant media and either continue-on with the minicircle maxiprep, or freeze pellet.

Generating minicircles from the parental cloning vector

To generate minicircles that are ready for transfection, you need your Minicircle Cloning Vector with your insert (gene, promoter-gene cassette, small RNA, etc.), SBI’s optimized, ready-to-transform ZYCY10P3S2T E. coli Minicircle Producer Strain (Cat.# MN900A-1), and arabinose (Cat.# MN850A-1).

Minicircles are produced from the full-sized Parental Minicircle using PhiC31 Integrase, which mediates a recombination event between the PhiC321 attB and attP sites on the parental plasmid (Figure 1). This reaction results in two products—the minicircle, which is now free from any bacterial DNA sequences—and the parental plasmid. To get rid of the parental plasmid, the I-SceI endonuclease recognizes and acts on the I-SceI sites on the parental plasmid, resulting in degradation of the parental plasmid.

Figure 1. Generating minicircle DNA from the Parental Minicircle Plasmid.

More about the ZYCY10P3S2T E. coli Minicircle Producer Strain The Minicircle Producer Strain harbors an arabinose-inducible system to express the PhiC31 integrase and the I-SceI endonuclease simultaneously. The ZYCY10P3S2T strain also contains a robust arabinose transporter LacY A177C gene.

Adding arabinose to the media turns on expression of the PhiC31 integrase and endonuclease genes, resulting in separation of the Parental Minicircle Plasmid into the individual minicircle and parental plasmids (from the PhiC31 Integrase activity), and the degradation of the parental plasmid (from I-SceI endonuclease activity).

Figure 1. Easy and clean minicircle production with the MC-Easy Minicircle DNA Production Kit. Note the genomic and parental DNA bands that only appear in the minicircle preps that did not use the MC-Easy Kit

Achieve sustained expression from minicircles after transfection in vitro and in vivo