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Following DNA purification, a Gibson assembly was performed in order to clone the 3xFlag- TRF1-FokI and 3xFlag-TRF1-FokI (D450) fragments into the doxycycline-inducible pCW57.1 lentiviral backbone. A GFP insert was also cloned into the final vector (Figure 2.1).

Briefly, the Gibson assembly is a method of molecular cloning in which multiple DNA fragments can be joined by using a single, isothermal reaction. The DNA fragments should contain around 20-40 base pair overlap with the adjacent DNA fragments, which is added by PCR. The Gibson assembly reaction involves the activity of three enzymes: an exonuclease, which digests the 5’ end of the DNA, resulting in single-stranded regions that the DNA fragments can anneal; a DNA polymerase, which adds nucleotides to fill in any gaps once the fragments have annealed; and a DNA ligase, which covalently joins the adjacent fragments (Gibson et al., 2009).

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Adding overlap regions into the fragments of interest by PCR

Primers were designed in order to amplify the inserts of interest out of the HFUW backbone by PCR. The resulting fragments will contain a region of overlap with the adjacent fragments of interest (i.e. GFP and pCW57.1). Overlap regions were also added to the pCW57.1 backbone and to the GFP insert by the same method. Primers were designed using Benchling.

The following was added to a PCR tube: 1μl 10X PfuUltra II Fusion HS DNA Polymerase (Agilent Technologies, 600670), 5μl 10X PfuUltra II reaction buffer + Mg2+_{, 2μl dNTP mix}

(10 mM), 1μl DNA template (10ng/μl stock), 3μl forward primer (5μM stock), 3μl reverse primer (5μM stock), and 35μl of RNase-free H2O.

The PCR reaction parameters were as follows: 1. 95°C for 1 minute

2. 95°C for 30 seconds 3. 60°C for 90 seconds 4. 72°C for 3 minutes

5. Repeat cycles 2 – 4 30 times 6. 72°C for 10 minutes

Gel electrophoresis for purification of PCR products

A 0.8% agarose gel was prepared by mixing 0.48g of agarose with 60 ml 1X TAE (Table 2.1). The mixture was microwaved until the agarose was dissolved. 1X SafeView Nucleic Acid Stain (NBS Biologicals, NBS-SV) was added to the gel mixture, which was then poured into a casting tray. Meanwhile, 10μl of 6X Gel Pilot® loading dye (Qiagen, 154015626) was added to 50μl of each DNA reaction mixture obtained from the PCR above. Once the gel had set, it was then transferred onto the electrophoresis tank, and 1X TAE was added until the gel was fully covered. 50μl of each DNA mixture was added to different wells, and a GeneRulerTM _{1kb Plus}

DNA Ladder (Sigma Aldrich, D0428-1VL) was also used. The gel was run at 120V for 1 hour. Following completion, the gels were analysed under a UV lamp, and the bands corresponding to the expected molecular weight of each fragment were cut out with a clean, sharp scalpel, and placed into a 1.5ml Eppendorf tube.

DNA extraction of purified PCR products

DNA fragments were extracted from the agarose gel using the QIAquick Gel Extraction Kit (Cat No. 28704), and the manufacturer’s protocol was followed. All centrifugation steps were

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carried out at 13,000 RPM in a conventional table-top microcentrifuge at 15-25°C. Briefly, the gel excised was weighed in an Eppendorf tube, and 3 volumes of Buffet QG was added to 1 volume of gel (100mg ≈ 100μl). The gel was incubated at 50°C for 10 minutes until it was completely dissolved, and the mixture was vortexed. For DNA fragments less than 500 bp or over 4 kb, 1 gel volume of isopropanol was added to the sample. A QIAquick spin column was placed in a 2ml collection tube, and the sample was added to the QIAquick column, followed by 1 minute centrifugation in order to bind the DNA to the column. The flow-through was discarded, and the QIA quick column was placed back in the collection tube. 0.5ml of Buffer QG was added to the QIAquick column and centrifuged for another 1 minute. The flow-through was discarded. Next, 0.75ml of Buffer PE was added to the column, and centrifuged again for 1 minute. The flow-through was discarded, and the dry column was centrifuged for an additional 1 minute. In order to elute the DNA, the QIAquick column was then placed in a clean 1.5ml microcentrifuge tube, and 30μl of H2O was added to the centre of the QIAquick

membrane. The column was left to stand for 1 minute, and then centrifuged for 1 minute. DNA concentration and purity were analysed using a Nanodrop® 1000 spectrophotometer (Thermo Scientific).

DNA Assembly Reaction (Gibson assembly)

Assembly of the DNA fragments obtained was performed by following the NEBuilder® HiFi DNA Assembly Reaction Protocol. Each reaction tube contained 20μl of mixture with the following:

- 10μl NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs, E2621L) - X μl of each DNA fragment (0.03-0.2 pmols) (The concentration of each fragment for

optimal assembly was calculated based on fragment length and weight. Each insert was added at a 2:1 insert: backbone ratio)

- Volume was completed to 20μl with H2O, if necessary

Samples were incubated in a thermocycler at 50°C for 60 minutes. Bacterial transformation

5μl of the above Gibson assembly products was added to 45μl of B100 E. coli competent cells, and mixed gently by flicking the tube 5 times. The mixture was placed on ice for 30 minutes, and then heat shocked at 42°C for 30 seconds. The tubes were placed on ice for 2 minutes, and 950μl of SOC outgrowth medium (Invitrogen, Cat. Number 15544-034) was added. The cells

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were incubated at 31°C for 60 minutes with shaking at 250RPM. Meanwhile, agar selection plates were warmed up to 31°C. The full volume of cells were streaked onto the agar plates, and incubated overnight at 31°C.

Plasmid expansion

Individual colonies were selected and grown overnight at 37°C in 5ml LB medium (Table 2.1) with Ampicillin (50ng/ml).

Plasmid isolation

Following expansion, plasmids were purified from B100 E. coli cells using the PureYieldTM

Plasmid Midiprep System (Promega), as described above. Plasmid analysis

Following plasmid purification, the plasmid DNA was digested with the XhoI and XbaI restriction enzymes to confirm that the Gibson assembly yielded the correct plasmids.