TITLE: MiniCircle Production
AUTHORS: Eric Danner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a cheap and efficent way to create Minicircles.</div><div class = "text-block"><span>This MiniCircle System was originally published: </span><a href="https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=21102455" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Nat Biotechnol. 2010 Dec; 28(12): 1287–1289.</span></a><span></span></div><div class = "text-block"><span>The cells and plasmid is available from SBI : </span><a href="https://www.systembio.com/wp-content/uploads/Minicircle-production-manual.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.systembio.com/wp-content/uploads/Minicircle-production-manual.pdf</span></a><span> . However the company kit requires you to recurently buy supplies. So we bought the kit once and then were able to make indefinite preps.</span></div><div class = "text-block"><span>This is our modifications drawn from from this paper :</span><a href="https://doi.org/10.1089/hum.2017.136" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1089/hum.2017.136</span></a><span>  . This requires the MC E.coli (ZYCY10P3S2T). This protocol requires you to make your bacteria chemically competent yourself and this protocol does not discuss how to make them.</span></div></div>

[STEPS]
?. [Clone Stuff into your minicircle backbone]
CloningThe backbone has a low-copy plasmid ORI. So you need to get a larger volume for mini-preps than usual. For sequencing one side of the multiple cloning sequence has a repetitive sequences for SecI cleavage and so can mess up the primer binding. You can Sanger Sequence from 5' end with : 5'-tgatggtcgagactcagcgg-3'   or from the 3' end with 5'-tcgccttcttgacgagttcttct-3'Use normal D5Halpha E-coli (or whatever you use) with Kan conditions for cloning as the ZYCY10P3S2T E.Coli strain transforms badly. Kan is toxic so requires 30-60 min shaking for resistance after heat-shock. Can use normal LB/kan media
?. [Re-transform into ZYCY10P3S2T]
Use 1-20ng of correct plasmid to re-transform into ZYCY10P3S2T cells. Preparation of these cells is the same as you would do for any chemically competent E.Coli strain.These cells can be grown in TB media overnight and then frozen in a 50% glycerol stock.
?. [Grow Culture]
Prepare your Terrific Broth Media. These cells can grown under LB/Kan but for MC production you should use TB. 1) in 900ml: 12g tryptone, 24g yeast, 4ml glycerol.   2) 10x phosphate buffer:  23.12g KH2PO4, 125.41g K2HPO4 brought to final vol 1L.   Autoclave both bottles and then mix (9:1). Check for precipitation in bottom after months.Add normal amount Kan (25-50ug/ml).
?. [Grow Culture]
Inoculate your bacteria/glycerol stab or picked colony in 2ml of TB for 2 hours. 37C, 250 rpm.Add 100ul to 200ml. Ideally pre-warmed TB w/Kan. Shake 220-250 rpm for 16 hr. (you don't want things to overgrow so check earlier than 16 hr).I have also just added a frozen glycerol stab to cold or warm TB/kan and things worked fine.
?. [Grow Culture]
Check OD600. The biggest issue to be careful of is bacteria overgrowth as it results in bacteria death and release of endotoxins, non-recombined Minicircle plasmid,s, gDNA.  You want an OD600 of 4-6. Look at the SBI protocol for more information. OD600 measurement (1:10 dilution with TB) -  1:10 dilution will give 0.4 - 0.6 which you then multiply by 10 to get original OD600. -If the OD600 >8.5 then throw out cells and do again also if pH is
?. [Grow Culture]
Prepare induction medium: 200ml LB +  200μl 20% arabinose (no kanamycin necessary). Mix 200ml induction media with 200ml culture.Check pH and titrate to about pH 7Now you may need to add NaOH to bring the pH to 7-7.3.
?. [Grow Culture]
MC induction: Take the 200ml culture + 200ml induction mix (check pH and adjust with 1N NaOH to 6.5 - 7) . Or 400ml induction media as noted above.Arabanose activates the recombinase and also activates a nuclease to destory the DNA sequence that has the portions of the plasmid we are not interested in.32 ° C, 250rpm, 3 - 5 hours. (no harm in letting it go to 5 hr)You can check with a mini (1-2ml) every hour to see the plasmid recombine. Mini prep is linearized with restriction enzyme and run on a gel. Lower band is recombined minicircle, upper band is entire plasmid.
?. [Grow Culture]
Pellet and Maxi: pellet the rest of the culture at 4 ° C and use Maxiprep (if necessary: ​​store the pellet at -20C)The plasmid is low copy, you can pellet up to 400ml (400 ml before the addition of the induction media) cells for OD600 = 6 in the MN Xtra Kit. In the maxi-kit it has a formula based on the OD600 and states how much lysis buffer to add. It is important to get enough lysis buffer to lyse all the cells. You do  not have to worry about having too much DNA binding the collumn, but rather that you dont get fullly lysed bacteria. Elute in 150ul TB
?. [Clean up MiniCircles]
linearize and run on gel. Take an aliquot of the purified MC and run undigested on a gel. You can also add a digested aliquot that is cut in one spot. You may see a smear at the very top showing the the bacteria gDNA. You will want this to get removed. Also you will likely see a band of the original plasmid size. This gives you an idea of how much plasmid/gDNA contamination you have.
?. [Clean up MiniCircles]
Cut plasmid backbone and gDNA with restriction enzymesNow we use restriction enzymes to cut up the plasmid that was not recombined in to a minicircle and also the gDNA.Find 2 restriction sites that exist in the backbone but not in your finished mini-circle.Digest: Final 200μl volume: 20μl buffer, 5μl Restriction enzyme 1, 5μl Restriction enzyme 2, total eluted maxiprep. Fill to 200 ul with H2O). Put on shaker 1-2h 37 ° C.
?. [Clean up MiniCircles]
Digest the linear DNA: Use plasmid-safe DNase to digest the linearized gDNA and plasmid. It will not digest the circular minicircle. 200 μl from restriction enzyme digestion + 25 μl 10x DNase buffe + 10 μl ATP + 5 μl H2O + 10 μl DNase. 37 ° C overnight (16 -20 hours) on shaker. Plasimid safe protocl
?. [Clean up MiniCircles]
Re-purifyHalt enzyme- 30min 70CUse pcr/gel cleanup mini kit. Each colum can bind 25g. So you might have to use multiple columns in serie. Elute in 20ul TE or water after warming column up to 70C. Check concentration with Nanodrop. Take an aliquot and run on a gel.