RF cloning protocol (modified from- Methods Mol Biol. 2014;1116:73-87):

*** That’s a universal protocol; adjust the PCR conditions to your specific project

Set up amplification reaction of the target gene(s) in 0.2 mL tubes, in a final volume of 50 μL, as follows (see Note 4):

1^st reaction assembly - Mega primer PCR

Final concentration	Volume/50 μL reaction	Stock solution	Component
0.4 ng/μL	2 μL	10 ng/μL	Target DNA
0.5 μM	1 μL	25 μM	Forward primer
0.5 μM	1 μL	25 μM	Reverse primer
200 μM each	1 μL	10 mM each	dNTPs mix
1×	10 μL	5×	Phusion HF Buffer
0.032 U/μL	0.8 μL	2 U/μL	Phusion DNA Polymerase
	34.2 μL		Ultrapure water

Mix the 0.2 mL tubes gently and spin briefly in a microcentrifuge.
Perform amplification reaction using the following parameters (see Note 4):

1^st reaction PCR program (change according to your specific project)

Cycles	Time (min:s)	Temperature (°C)	Step
1	01:00	95	Denaturation
25	00:30	95	Denaturation
	01:00	60	Annealing
	01:30	72	Elongation
1	06:00	72	Elongation
	∞	10	Cooling

Following completion of the amplification reaction analyzes aliquots (2–3 μL) from each reaction on a 1 % agarose gel (see Note 5).
Purify the PCR product(s) (see Note 6) and measure DNA concentration using a NanoDrop spectrophotometer.
Assemble the PCR products (mega-primers) and the destination vector in 0.2 mL tubes, in a final volume of 50 μL, as follows (see Note 7):

	2^nd reaction assembly – Plasmid annealing

Final concentration	Volume/50 μL reaction	Stock solution	Component
0.2 ng/μL	1 μL	10 ng/μL	Destination plasmid
2 ng/μL	X μL	X ng/μL	PCR product I
2 ng/μL	X μL	X ng/μL	PCR product II (optional)
200 μM each	1 μL	10 mM each	dNTPs mix
1×	10 μL	5×	Phusion HF Buffer
0.032 U/μL	0.8 μL	2U/μL	Phusion DNA Polymerase
	Complete to 50 μL		Ultrapure water

Mix the 0.2 mL tubes gently and spin briefly in a microcentrifuge.
Perform the assembly reaction using the following parameters (see Note 8):

2^nd reaction PCR program - Insert-plasmid reaction

Cycles	Time (min:s)	Temperature (°C)	Step
1	00:30	95	Denaturation
30	00:30	95	Denaturation
	01:00	60	Annealing
	05:00	72	Elongation
1	07:00	72	Elongation
	∞	10	Cooling

Following completion of the amplification reaction analyzes aliquots (10 μL) from each reaction on a 1 % agarose gel (see Note 9).
Transfer 10 μL from the RF reactions into a new 1.5 mL tube. Add 1 μL DpnI, spin briefly, and incubate for 1–2 h at 37 °C.
Spin briefly the DpnI-treated reaction using a microcentrifuge.
Transform the DpnI-treated reaction into competent DH5α E. coli cells.
Add 1–10 μL of reaction into 90–100 μL competent cells (see Note 10). Mix gently.

Incubate on ice for 30–60 min. Heat the tube at 42 °C for 1 min and return to ice. Add 1 mL of LB and incubate with shaking for 1 h at 37 °C.

Plate transformed mix on LB agar plates with the appropriate antibiotic.
Incubate plates overnight in a 37 °C incubator.
On the following day, select 6-10 single colonies from the transformation plate, and perform colony PCR to identify positive colonies as described below (see Note 11). If no colonies are observed on the plate consults Note 12 for details on how to proceed.
Set up PCRs in 0.2 mL tube, in a final volume of 20 μL, as follows (see Note 13)

		Colony PCR reaction

Final concentration	Volume/20 μL reaction	Stock solution	Component
1.25 μM	1 μL	25 μM	Forward primer
1.25 μM	1 μL	25 μM	Reverse primer
1x	10 μL	2x	Master mix
	8 μL		Ultrapure water

18. Using a sterile toothpick select single colonies and transfer the bacteria into the labeled 0.2 mL PCR tubes. Swirl well.

19. Using the same toothpick maintain each clone by inoculating liquid or plate containing the appropriate antibiotic (see Note 11).

20. Mix gently the 0.2 mL PCR tubes and spin briefly.

21. Perform colony PCR using the following parameters (see Note 13):

Cycles	Time (min:s)	Temperature (°C)	Step
1	01:00	95	Denaturation
25	00:30	95	Denaturation
	01:00	60	Annealing
	01:30	72	Elongation
1	06:00	72	Elongation
	∞	10	Cooling

Analyze PCR products on 1 % agarose gel to verify that the DNA product obtained is of the correct size. Select 2–3 individual positive colonies (see Note 14).
Inoculate positive clones into 10 mL LB medium supplemented with antibiotic in 50 mL conical tubes. Concentration of the antibiotic depends on the type of the antibiotic used.
Grow bacteria with shaking at 37 °C, for 16–20 h.
Harvest cells and extract DNA using a plasmid DNA purification kit (see Note 15).
Measure DNA concentration using a NanoDrop spectrophotometer.
Send the 2–3 positive selected clones for DNA sequencing.

Notes

Primers (forward and reverse) for the RF reaction include at the 5′-end a vector-specific sequence, complementary to the site of integration into the recipient vector, and at the 3′-end a sequence complementary to the gene of interest used for amplification of the gene. The total primer length is 50–60 nt. The length of the overlapping sequence to the destination vector can range from 20 to 40 nt with the recommended length of 30 nt. The length of the gene-specific sequence is variable and should be designed to satisfy the melting temperature ( Tm ) requirements. The Tm for the gene-specific sequence should be between 60 and 70 °C, where A or T, and G or C, each contributes to the Tm 2 and 4 °C, respectively. Synthetic primers were ordered from Integrated DNA Technologies (IDT; Leuven, Belgium) or Sigma-Genosys (Rehovot, Israel). Primers up to 60 nt are ordered with only basic desalting purification. Longer primers are purified either by HPLC or by SDS-PAGE.

We found that preparation of competent cells according to the procedure previously described is best suited for the high efficiency of the RF reaction. In this procedure, cells are grown at 18 °C prior to harvesting and preparation of the competent cells. 90–100 μL of competent cells are used for transformation of 1–10 μL of DpnI-treated RF reaction.

On a routine basis we use plasmid DNA obtained from various sources as the template for amplification of the gene of interest. However, we have also used other sources for amplification such as chromosomal DNA from various microorganisms and cDNA libraries. When using DNA sources other than plasmid, optimization of the PCR amplification might be essential ( see Note 4 ).

The components and the reaction conditions for the PCR amplification can be modified if needed. Addition of dimethyl sulfoxide (DMSO), 5–10 % v / v, might be essential for successful reaction if the target DNA has high GC content. Performing a gradient PCR or changing the annealing temperature might be essential, in certain cases, to obtain successful amplification. If large DNA fragments are amplified increasing the elongation time might be essential.

Analyzing the PCR amplification reactions on agarose gel is essential step to ensure that the correct size PCR product was obtained as the major band. If no PCR product was obtained, reexamine the primer design. In addition, make sure that source DNA used is correct and if needed alter the PCR conditions (see Note 4).

We highly recommend purifying the PCR product(s) directly from the PCR mix and not from the agarose gel. We have noticed that purifying the PCR product following extraction

from the agarose gel resulted in loss of much of the DNA and reduced yield of gene integration into the destination vector in subsequent steps. Removal of the PCR product from an agarosegel is an optional step when multiple bands are obtained in the initial PCR amplification step.

In certain cases altering the components for the integration step might be essential. If addition of dimethyl sulfoxide (DMSO) was essential for PCR amplification in the fi rst stage ( see Note 4 ), it should be used in this stage, as well. On a routine basis, we used 20 ng from the destination vector and 100 ng from each PCR product (mega-primers). However, increasing the amount of the mega-primers (up to 250–300 ng per reaction) might improve, in certain cases, the efficiency of integration. If more than two PCR products (as specified in the protocol) are used for simultaneous integration into the destination vector use the same DNA concentration for the additional mega-primers.

If the total size of the newly synthesized vector (DNA insert(s) plus destination vector) is large (>15 kb) increasing the elongation time might be essential. The recommended time for the elongation step for the Phusion DNA polymerase is 15–30 s/kb. In addition, optimization of the integration reaction using gradient PCR might be essential, primarily when multiple mega-primers with various length and complexity are used simultaneously.

Analyzing the RF reaction on agarose gel is an optional, but highly recommended, step. The analysis should give a strong indication on how successful the assembly reaction worked. If high molecular band, corresponding to the newly synthesized plasmid, is observed , it is highly likely that the reaction was successful. We recommend on proceeding to DpnI treatment followed by transformation even if no upper band is observed. We have observed in many cases that the reaction worked well even if no clear upper band was detected following the agarose gel electrophoresis analysis.

10.

The amount of the DpnI-treated RF reactions used for transformation varies between 1 and 10 μL. If we observed an intense high molecular DNA band corresponding to the newly synthesized plasmid following the agarose gel analysis only 1–2 μL are sufficient to obtain sample but distinct colonies for subsequent analysis. However, if no upper DNA band is observed, it is recommended to use the entire 10 μL DpnI-treated reaction for transformation.

11.

Before selecting clones for DNA sequencing it is advisable to perform colony PCR to ensure that the selected clones harbor the target genes. The colony PCRs are carried out using forward and reverse primers, derived from sequences flanking the cloning sites in the destination vector. Alternatively, a combination of a specific primer from the insert and a primer from the destination vector is used. It is advisable to perform a negative control PCR where the destination vector without the target gene is used. Before performing the PCR, it is essential to maintain the selected clones by striking on a selective plate or into a liquid media. For multiple DNA fragments integration at distinct positions, two sets of primers are used for amplification of the same colony. Alternatively, only the two most distal primers are used for identification of clones in which the two DNA fragments are present.

12.

The absence of colonies following transformation step is a clear indication for a failure either at the DNA assembly stage or in the transformation procedure. In order to identify the source of failure follow the following guidelines: Remove 1–2 μL from the RF reaction and perform PCR analysis as described in Subheading 3.2 , using flanking primers. Analyze PCR on agarose gel.

● If a PCR product at the expected size is observed, the RF reaction was successful. In this case, the transformation stage has failed. Reexamine the effi ciency of the competent cells (see Note 2) and make sure that the antibiotic used for selection was correct. An additional possibility for failure is that the gene product cloned is toxic to the bacterial cells and its expression, even at very low levels, is detrimental to cell growth. In this case, addition of 1 % glucose to re-cloning of your gene into a tighter expression system may be required to obtain transformants following the cloning stage.

● If no PCR product or PCR product corresponding to the empty vector was obtained it is likely that the RF integration reaction has failed. In this case reexamine the primers designed for the RF cloning. If the primer design is correct, it is likely that the reaction conditions should be changed ( see Notes 7 and 8 ). Repeat the RF reaction with freshly prepared components. Make sure to re-dilute the destination plasmid from a verifi ed stock solution.

● When several DNA fragments are used for simultaneous integration, it is possible that not all new clones will harbor all the different DNAs. If only partial integration is observed the reaction conditions should be changed. We have observed that changing the annealing temperature in the RF reaction can have a positive effect on multiple DNA fragments assembly.

13.

When multiple colony PCRs are performed in parallel it is recommended not to pipette each of the reaction components separately into each PCR tube. In this case a solution mix including all the reaction components can be assembled for the number of reactions needed. Mix the solution and transfer 20 μL aliquots into the 0.2 mL PCR tubes and proceed with inoculation of the bacterial colonies. If needed, PCR components and or reaction conditions should be optimized to adjust the target gene amplifi ed ( see Note 4 ).

14.

If primers fl anking the target gene are used for colony PCR analysis, the PCR products observed from positive clones should run at a higher molecular weight compared to that of

the parental plasmid.

15.

It is recommended to prepare a glycerol stock from the selected clones (final glycerol concentration 20–25 % v/v). The stock should be stored at −80 °C until used. Avoid freezing–thawing cycles when using −80 °C glycerol stock.