OriCiro® Cell-Free Cloning System/PASS V4.1.2

Thaw 2X RA Mix on ice , mix it well with a vortex mixer at a maximum speed and spin down with a micro-centrifuge.

Prepare the following mixture on ice, and mix well by pipetting. (*1)

Sample

A	B
Nuclease-Free Water	2.5 - X μl
DNA Fragments (*2) (up to 20 ng as total fragments)	X μl
2X RA Mix	2.5 μl
Total	5 μl

Control

A	B
Nuclease-Free Water	0.5 μl
oriC Cassette (50 pg/μl)	1 μl
Control Fragment (1 ng/μl)	1 μl
2X RA Mix	2.5 μl
Total	5 μl

(*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.

**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.

*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).

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.

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.

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

A	B
< Amp pre-mixture>	x1 reaction (*2)
Nuclease-Free Water	4μl
5X Buffer I	2μl
5X Buffer II --> Vortex Mixing	2μl
10X RE Mix --> Pipette mixing (*3)	1μl
Total	9μl

(*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.

(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.

**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.

(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.

*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.