TITLE: OriCiro® Cell-Free Cloning System/PASS V4.1.2
AUTHORS: shigemasa.s  

[DESCRIPTION]
OriCiro® Cell-Free Cloning System is a rapid and powerful tool replacing cumbersome DNA cloning 
(plasmid construction) process relying on E. coli. The system consists of two kits. OriCiro Assembly Kit allows seamless assembly of multiple overlapping DNA fragments. The assembly product can be added directly to OriCiro Amp Kit to get selective amplification of your target circular DNA (Figure 1). The amplified product is supercoiled DNA topologically identical to plasmid DNA isolated from E. coli. 

  
1. OriCiro Assembly Kit: Multiple DNA fragments are assembled seamlessly at 42 ̊C for 30 minutes via ~40 bp overlapping ends (Figure 2). DNA fragments generated by PCR or restriction enzyme digestion are available. Our unique enzyme-based annealing mechanism allows powerful assembly up to 50 fragments simultaneously. 

  
2. OriCiro Amp Kit: The reaction consists of 26 purified enzymes involved in chromosome replication of E. coli. The chromosome replication cycle repeats autonomously at around 30 ̊C, enabling exponential amplification of circular DNA having oriC with extremely high fidelity (10-8 error/base/cycle) (Figure 3). The kit yields up to 1 μg circular DNA per 10 μL reaction at 33 ̊C for 6 hr. The maximum amplification size is 50 kb in the current version of the kit. 
n.b. OriCiro Amp NEEDS oriC Cassette (0.4 kb) which can be inserted into circular DNA using OriCiro assembly kit. 

  
References: 
1. T. Mukai, T. Yoneji, K. Yamada, H. Fujita, S. Nara, M. Su'etsugu, Overcoming the Challenges of Megabase-Sized Plasmid Construction in Escherichia coli, ACS Synthetic Biology, 2020, 9 (6), 1315- 1327 
2. T. Hasebe, K. Narita, S. Hidaka, M. Su'etsugu, Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction. Life, 2018, 8 (43) 
3. M. Su’etsugu, H. Takada, T. Katayama, H. Tsujimoto, Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle, Nucleic Acids Research, 2017, 45 (20), 11525– 11534 






[GUIDELINES]
a. Design and preparation of DNA fragments 
Each end of DNA fragments must have overlapping sequences which is typically introduced by PCR using tailed primers. For assembly of a smaller number of fragments, shorter overlap (~25 bp) is enough. Longer overlap (40-60 bp) can improve the assembly specificity and is particularly required for assembly of a larger number of fragments (>10 fragments) or larger sized DNA (>10 kb). Tm value does not have to be considered. The overlap ~10 bp away from the fragment end is acceptable. Restriction digested fragments with 5’ or 3’ sticky end are also available. 
  
b. Design and Preparation of your oriC cassette 
The oriC fragment having overlapping ends against your target fragments can be prepared by PCR using oriC Cassette (included in this kit) as a template, and primer pairs containing ~40 bp overlap sequences at their 5’tails. 

Typical primer sequence to amplify your oriC Cassette will be as follows:
Forward primer:5’-[~40 nt overlap for tail of your DNA fragment] +CTGCTCTGATGCCGCATAG-3′
Reverse primer: 5’-[~40 nt overlap for head of your DNA fragment (Reverse)] +GTGTCGGGGCTGGCTTAAC-3' 

 
c. DNA quantity calculation for OriCiro Assembly Kit 
Each DNA fragment should be added at equal molar ratio. The applicable amount of DNA fragments in the 5 μl assembly reaction is 1 pg - 20 ng as total fragments. Total 20 ng DNA is optimal for more than 10 fragments assembly. The amount of each DNA fragment is calculated by the following formula. 
                    [Fragment size] / [Total assembly size] × [Total DNA amount (1 pg – 20 ng)] 
As an example of assembly for two fragments; “oriC Cassette (0.4 kb)” and “Control Fragment (7.5 kb)”, where the “total assembly size” is 8 kb, the amount of each fragment should be as follows. 
                    oriC Cassette: 0.4 kb / 8 kb × 1 ng = 50 pg 
                    ControlFragment:7.5kb/8kb×1ng ≈ 1ng 
The equal molar ratio is important for the assembly of a larger number of fragments (>10 fragments). In this case, we recommend use of DNA fragments with approximately equal length, whose concentrations are quantified precisely using fluorescence-based method (e.g., Q-bit, Thermo Fisher Scientific) or Agilent 2100 Bioanalyzer (Agilent Technologies). 

d. oriC Cassette (378 bp)
     
e. Control Fragment (7.5 kb) 

Control Fragment was constructed by PCR amplification of the whole pBeloBAC11 plasmid using the following primers: 5’- CTATGCGGCATCAGAGCAG -3’ and 5’- GTTAAGCCAGCCCCGACAC -3’. The oriC Cassette will be inserted into a site downstream of the lacZa gene of pBeloBAC11. The resultant plasmid has oriC, BAC origin and a chloramphenicol-resistant gene, and is able to maintain the plasmid in E. coli after transformation. 



  



[STEPS]
SECTION: Assembly Reaction
1. Thaw 2X RA Mix on ice, mix it well with a vortex mixer at a maximum speed and spin down with a micro-centrifuge. 
SECTION: Amplification Reaction
5. Turn on a thermal cycler or an air incubator and preheat at 33°C. 

Avoid evaporation of the reaction during incubation. If the thermal cycler is used, its lid should be set at 40°C. 
SECTION: Amplification Reaction
6. After 5X Buffer I and 5X Buffer II are thawed on ice, mix well with a vortex mixer and spin down with a microcentrifuge. After 10X RE Mix is thawed on ice, mix gently with the vortex mixer and spin down with the microcentrifuge. 




SECTION: Amplification Reaction
7. Prepare the following pre-mixture on ice. Mix before and after the addition of 10X RE Mix as indicated. *1 


 

(*1): Use a 0.2ml PCR tube to avoid evaporation
(*2): Amp premixture for multiple reactions can be prepared as a single “master mix” by multiplying the volume of each reagent by the number of reactions. 
(*3): For the pipette mixing, set pipette volume to the total mixture volume, and pipetting up and down four times with agitation. 




SECTION: Assembly Reaction
2. Prepare the following mixture on ice, and mix well by pipetting. (*1) 

Sample 
 
Control 
 
(*1): 
• Use a 0.2 ml PCR tube to avoid evaporation. 
• Vortex mixing is not recommended because of small volume of viscous sample. 
• For the pipette mixing, set pipette volume to the total mixture volume, and pipetting up and down four times with agitation. 

 (*2): 
• Include your intended oriC Cassette for the subsequent amplification reaction. 
• Each DNA fragment should be added at equal molar ratio. The applicable quantity of DNA fragments is 1 pg - 20 ng. The optimal quantity for more than 10 fragments assembly is 20 ng as total fragments. (see Appendix c. for the quantity calculation). 
• DNA fragment dissolved in TE buffer is acceptable. 
SECTION: Assembly Reaction
3.  Incubate the mixture at 42°C (*3) for 30 minutes and hold on ice before use.(*4) 
 (*3):  Needs 30-42°C 
 (*4):  Although the assembly product can be stored at -4°C for a few days before the subsequent amplification step, i  mmediate use is recommended for best results.
SECTION: Assembly Reaction
4. Optional Step (*5): Heat Treatment 


Immediately after the 42°C incubation in step (3), transfer the mixture to 65°C block and heat it for 2 minutes, followed by quick cooling on ice. 

(*5): “Heat treatment” can eliminate mis-assembly by products, and is recommended for the best results particularly when you intend to assembly a large number of fragments (over five fragments). 




SECTION: Amplification Reaction
8. (Optional) Pre-incubation (*4):

Incubate the Amp pre-mixture at 33°C for 15 minutes. 

(*4): “Pre-incubation” option stimulates an initial stage of the amplification to allow stable amplification of the circular DNA particularly when the amplification is difficult due to a low amount of the template DNA molecules. 



 
SECTION: Amplification Reaction
9. Add 1 μl of the assembly product (or oriC circular DNA), and mix with pipetting (*3). Incubate the mixture at 33°C for 6 hours(*5) and hold at 12'C (*6) or on ice before use. 
(*5): 
 The incubation time can be shortened to 3 hours particularly when already supercoiled DNA is used as a template. The 6 hours incubation allows stable amplification particularly of the assembly product which requires gap-repair process. 
Higher temperature up to 40'C or longer incubation up to 16 hours is acceptable, but tends to produce other short DNA byproduct than your target. 
(*6): 
A thermal cycler program is useful to hold automatically at 12°C after the 33°C incubation. 





SECTION: Amplification Reaction
10. (Optional) Finalization (*7):

Dilute the reaction of step (9) two times with 1X Amp Buffer (*8) and further incubate at 33°C for 30 minutes. 

(*7): When replication intermediates (open circular or catenane DNA etc.) is abundant, “Finalization” option can convert them to supercoiled DNA. 
(*8): 1X Amp Buffer is prepared by mixing 5X Buffer I and 5X Buffer II to final 1X concentration with Nuclease-Free Water. Extra volumes of the 5X Buffers are provided for this option. 






SECTION: Amplification Reaction
11. Check the amplified products using agarose gel electrophoresis.(*9)

(*9):
• Typical DNA concentration before Finalization option is 50-100 ng/μl 
• The gel-loading buffer should contain SDS etc. to remove proteins from DNA. 
• Because the product is supercoiled form, Supercoiled DNA Ladder (New England Biolabs) is recommended as a size maker. Alternatively, analyze it by restriction mapping. 

The products can be stored at 4°C for several days. For long-term storage, add final 20 mM EDTA before storage at -20°C. Alternatively, purify the product with phenol/chloroform, followed by ethanol precipitation.