MOLECULAR ANALYSES

Select approximately 20 nucleotides both upstream and downstream of the core region of each att site formed in the reporter plasmid after recombination takes place.

Use an online oligo design tool to define the best forward primers annealing to promoter sequence and reverse primers annealing to terminator sequence present in the reporter plasmid.

Define oligo pairs to obtain two amplicons for each reporter plasmid.

The primers used in our studies are presented in Table 7 .

A	B	C	D
Oligonucleotides used for amplification of Amplicon I and sequencing of attL sites
Promoter	Forward primer (5’ -> 3’)	nt	Model
EFa_966F	TTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTG	35	Mammal
35S_282F	ATTGATGTGATATCTCCACTGACGTAAGGGATGACGCAC	39	Plant
attR	Reverse primer (5’-> 3’)	nt	Model
attR _Int2_R	GTGTCTACGCGAGATTCTCGCCGGACCGTCGACATACTGC	40	All models used
attR _Int4_R	AGTTTTCAACCCTTGATTTGAATAAGACTGCTGCTTGTGT	40
attR _Int5_R	ATAACTCTCCTGGGAGCGCTACACGCTGTGGCTG	34
attR _Int7_R	CTGTGTGAGAGTTAAGTTTACATGGGCAAAGTTGATGAC	39
attR _Int9_R	TGGAAGTGTGTATCAGGTAACTGGATACCTCATC	34
attR _Int13_R	GTAGAACTTGACCAGTTGGTCCTGTAAATATAAGCAATCC	40
attR _phiC_R	CCAACTGGGGTAACCTTTGGGCTCC	25
attR _Bxb1_R	CTGGTCAACCACCGCGGTCTCCGTCGTCAGGATC	34
Oligonucleotides used for amplification of Amplicon II and sequencing of attR sites
attL	Forward primer (5’-> 3’)	nt	Model
attL_Int2_F	GGAGTAGCTCTTCGCCCGAGAACTTCTGCAAG	32	All models used
attL_Int4_F	CGACCTGAAATTTGAATTAGCGGTCAAATAATTTGTA	37
attL_Int5_F	GACGGCCTGGGAGCGTTGACAACTTGCGCACC	32
attL_Int7_F	GTCCGTCTGGGTCAGTTGCCTAACCTTAACTTTTAC	36
attL_Int9_F	ATAATTGGCGAACGAGGTATCTGCATAGTTATTCCGAAC	39
attL_Int13_F	TCCAGATCCAGTTGTTTTAGTAACATAAATACA	33
attL_phiC_F	TGCCAGGGCGTGCCCTTGAGTTCTCTCAGT	30
attL_Bxb1_F	TGTCGACGACGGCGGTCTCAGTGGTGTACGGT	32
Terminator	Reverse primer (5’ -> 3’)	nt	Model
TermiAni_205R	AATGATTTGCCCTCCCATATGTCCTTCCGAGTG	33	Mammal
NOSt_283R	ATAACAATTTCACACAGGAAACAGCTATGACATGATTACG	40	Plant

Use a high-fidelity polymerase with non-template–dependent terminal transferase activity to insert a deoxyadenosine and the ends of generated amplicons.

Prepare a PCR mix for all reactions plus one (n+1) to account for pipetting errors. Include a negative control with water instead of DNA; positive control will require a previous synthesis of the expected recombined reporter plasmid.

Combine the reagents in the order shown below in Table 8, mix well by vortexing and spin briefly:

TABLE 8. PCR reaction mix components

A	B
Component	Volume to add (µl)
dH2O nuclease free	18.65
Buffer 10x	2.5
MgCl2 [50 mM]	1.5
PCR Fw primer [10 μM]	0.75
PCR Rev primer [10 μM]	0.75
dNTP [10 mM]	0.75
Taq DNA polymerase	0.1

Add 24.5µL of the PCR mix to 0.2 mL PCR tubes..

To each respective tube, add 20ngof template DNA and adjust the final volume to 25 µl if the DNA is too concentrated.

Gently pipette each sample up and down ten times to mix thoroughly. Place the PCR microtubes into a thermal cycler, and run the following program listed in Table 9 (volume = 25 μL).

TABLE 9. PCR cycling condition

A	B	C	D
Cycle no.	Denature	Anneal	Extend
1	94°C, 3min
2-34	94°C, 30s	65°C, 30s	72°C, 60s
35			72°C, 5min

Resolve amplicons by electrophoresis in agarose gel following PCR. Run settings and gel density will depend on amplicon size according to the analyzed gene length and oligo pairs used.

Excise the amplicon bands by cutting a square around them with the help of a scalpel on a UV light or blue light transilluminator.

Proceed with amplicon purification using commercial DNA Clean-Up and Concentration kits, following the manufacturer’s recommendations.

Clone purified amplicons in an entry vector to ensure high-quality sequencing results. Although specifics may vary depending on the plasmid, we recommend a molar ratio of 1:3 (vector to amplicon) and 1.5 U of T4 ligase in 5 µl reactions with incubation at 16°C.

DH10b chemically competent cells were transformed with ligation products.

Add 5µL of the ligation reaction to 200µL of cells thawed On ice.

Incubate cells On ice for 0h 30m 0s.

Subject cells to heat shock at 42°Cfor 0h 0m 45s and return to ice for 0h 2m 0s.

Add 1mL of LB or SOC medium.

Incubate at 37°C for 1h 0m 0s and then plate different dilutions on LB plates with appropriate selecting agents. Incubate 1h 0m 0s at 37°C.

Screening for positive transformants by colony PCR. A polymerase with less fidelity can be used in this step. Combine the reagents in the order listed in Table 10 below, mix well by vortexing and spin briefly:

TABLE 10. PCR reaction mix components for colony screening.

A	B
Component	Volume to add (µl)
dH2O nuclease free	18.65
Buffer 10x	2.5
MgCl2 [50 mM]	1.5
PCR Fw primer [10 μM]	0.75
PCR Rev primer [10 μM]	0.75
dNTP [10 mM]	0.75
Taq DNA polymerase	0.1

Add 25µL of the PCR mix to 0.2 mL PCR tubes.

With a sterile toothpick or 200 µl pipetting tip, pick approximately 1/3 of each colony and add it to their respective tubes containing the PCR mix.

Gently pipette each sample up and down ten times to mix thoroughly. Place the PCR microtubes into a thermal cycler, and run the following program listed in Table 11 (volume = 25 μL)

TABLE 11. PCR cycling condition for colony screening

A	B	C	D
Cycle no.	Denature	Anneal	Extend
1	94°C, 10min
2-34	94°C, 30s	60°C, 30s	72°C, 90s
35			72°C, 5min

Resolve amplicons by electrophoresis in agarose gel following PCR.

Select multiple confirmed clones to isolate plasmids using commercial kits following the manufacturer’s recommendations and have the purified plasmids sequenced.

Analyze sequencing electropherograms and alignment to expected sequences to confirm proper DNA recombination by Integrase activity.

TABLE 12. Troubleshooting for the molecular analyses stage

A	B	C	D
Step	Problem	Possible reason	Solution
10	Unspecific amplification and unexpected bands on agarose gel	Oligos annealing at att sites has a 3’ end complementarity to both original and recombined att sites	Increase the annealing temperature to more sselective conditions