An E. coli culture containing the pGBKT7 vector was grown at 37°C with shaking overnight in the presence of ampicillin. Plasmid DNA was isolated using the Wizard Plus SV Minipreps DNA Purification System (Promega) and then quantified. The vector was linearized via restriction digest with BamHI and SalI for one hour at 37°C, according to the manufacturer's instructions.
The fragment of interest (AnkH) was amplified from Legionella pneumophila strain AA100 with primers containing cloning ends compatible with the linear vector and necessary for In‑Fusion cloning. Primers were designed using Takara Bio's online primer design tool. The PCR reaction was set up as shown below:
| 12.5 µl |
CloneAmp HiFi PCR Premix |
| 1.5 µl |
Forward primer |
| 1.5 µl |
Reverse primer |
| 5 µl |
DNA template |
| 4.5 µl |
Nuclease-free water |
|
| 25 µl |
Total volume |
PCR was performed with denaturation at 98°C x 10 sec, annealing at 55°C x 15 sec, and extension at 72°C x 1 min, for a total of 35 cycles.
The PCR product was run on a 1% agarose gel, purified with the provided Macherey-Nagel Nucleospin Gel and PCR Clean-Up kit, and then quantified. In-Fusion Cloning reactions, including positive and negative controls, were set up as recommended in the user manual (see table below). The insert and linearized vector for the positive control reaction were both provided with the In‑Fusion HD Cloning Plus system (Note: In-Fusion HD Cloning Plus has been discontinued and replaced with In-Fusion Snap Assembly)
| In-Fusion Cloning reaction setup |
| Component |
AnkH experiment |
Positive control |
Negative control |
5X In-Fusion HD Cloning
Plus Enzyme Premix |
4 µl |
2 µl |
2 µl |
| Linearized vector |
4 µl
(pGBKT7 +
BamHI + SalI) |
2 µl
(pUC19 Control
Vector) |
4 µl
(pGBKT7 +
BamHI + SalI) |
| Insert |
5 µl
(AnkH PCR product) |
1 µl
(Control insert) |
0 µl |
| Water |
7 µl |
5 µl |
4 µl |
| Total volume |
20 µl |
10 µl |
10 µl |
In-Fusion Cloning reactions were incubated for 15 min at 50°C. The provided Stellar Competent Cells were transformed with the reaction mixtures. Transformations were plated on LB plates containing 100 µg/mg ampicillin and incubated overnight at 37°C. Colonies were picked for overnight cultures in LB + ampicillin media and grown overnight at 37°C with shaking at 250 rpm. Plasmid DNA was isolated from overnight cultures using the Wizard Plus SV Minipreps DNA Purification System. PCR was then used to determine the presence of the insert in each clone. Both positive and negative experiments produced the expected results. Ten out of ten experimental colonies were confirmed to contain the AnkH fragment.
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