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After overnight incubation, distinct single colonies were selected for analysis by PCR to determine if the ligation had been successful. PCR was carried out at the conditions described in section 2.2.5. Each 20µl reaction contained the following: transformed bacterial clone DNA (achieved by inoculating the PCR tube with a pipette tip

containing each bacterial colony), 2µl of 10x FastStart High Fidelity reaction buffer with 18mM MgCl2, 0.4µl PCR grade nucleotide mix (200nM final concentration of

each dNTP), 0.4µl DMSO, 13µL PCR grade water, 0.2µL FastStart High Fidelity Enzyme Blend (5U/µl), 2µl of both sense and anti-sense primers (400nM final

concentration; see table 4.1 for primer sequences). Ten colonies were selected for each ligation reaction (SLC22A6 and SLC22A8). Negative control reactions were also carried out in the absence of bacterial DNA to check for PCR reagent contamination. At the same time, aliquots of 3ml LB broth (supplemented with 50µg/ml ampicillin) were inoculated with each of the 10 colonies selected from each ligation reaction. Any colonies in which the SLC22A6 or SLC22A8 template could be amplified by PCR were subsequently grown for 8 hours in the culture to allow the transformed bacteria to reach exponential growth rate. The 8 hour cultures were then transferred to a larger culture (15ml LB broth supplemented with 50µg/ml ampicillin) and grown overnight at 370C with vigorous shaking (300rpm; ThermoScientific Thermo Max Q 4450).

The following day, SLC22A6 and SLC22A8 template positive bacterial colonies grown overnight were further analysed by restriction digest. The plasmid DNA of 8 colonies from which SLC22A6 and SLC22A8 had been successfully amplified (figure 4.3) was first extracted as described in section 4.2.2 before a 2 hour restriction digest reaction. The AleI and EcoRV restriction endonucleases were used as both the SLC22A6 and SLC22A8 plasmid DNA contains one AleI and one EcoRV restriction site in the insert and vector section of the sequence respectively. The 50µl reactions were set up in 200µl low density plastic PCR tubes and incubated at 370C in a 96 well block thermo cycler for 2 hours. Reaction mixtures contained the following components, made up to 50µl with water: 2µg plasmid DNA, 10 units (AleI; 1µl, EcoRV; 0.1µL) of restriction endonuclease, 5µl (1 x final concentration) of appropriate buffer (NEB buffer 4 for AleI reactions, NEB buffer 3 for EcoRV reactions), 0.5µl (1µg/ml final concentration) BSA

104 (EcoRV reactions only). Resulting DNA was visualised by gel electrophoresis (see section 2.2.4). Plasmid DNA containing the successfully ligated vector, positive for the SLC22A6 or SLC22A8 template, was linearised by digestion with both AleI and EcoRV, with successfully linearised DNA traveling less far on an agarose gel than its respective supercoiled form (figures 4.4-4.5).

Three of the clones shown to express the ligated plasmid and therefore SLC22A6 or SLC22A8 template were sent to Source Bioscience Lifesciences (Cambridge, UK) for single tube sequencing. Linear plasmid DNA at a concentration of 100ng/µl was supplied and the plasmid DNA sequenced from the T7 promoter and BGH reverse priming sites within the pcdna3.1/V5-His TOPO vector sequence; this enabled the full insertion site of the vector to be sequenced. Sequence results were then analysed to ensure the inserted SLC22A6 or SLC22A8 template was identical to the published protein coding sequence for each gene. A single clone for SLC22A6 and SLC22A8 was then selected for subsequent transfection.

105 Figure 4.3: Visualisation of successfully amplified SLC22A6(A) and SLC22A8(B) template DNA from plasmid DNA of transformed E.coli cultures. Samples were run on a 1% agarose gel using NEB 10kb ladder as a reference. From left to right: 1kb DNA ladder (1), positive control (template amplified from cDNA clone; (2)), SLC22A6(A) or SLC22A8(B) template DNA amplified from plasmid DNA (3-10), negative control reaction containing no DNA (11). SLC22A6 template DNA was successfully amplified from plasmid DNA in all 8 colonies of transformed E.coli (part A, lanes 3-10).

SLC22A8 template DNA was successfully amplified from plasmid DNA in 6 of the 8 colonies of transformed E.coli included (part B, lanes 5-10).

106 Figure 4.4: Restriction digest of SLC22A6 positive plasmid DNA from transformed E.coli cultures with AleI (A) and EcoRV (B). Samples were run on a 1% agarose gel using NEB 10kb ladder as reference. From left to right: 1kb DNA ladder (1),

supercoiled SLC22A6 cDNA clone as a positive control (2), supercoiled SLC22A6 positive plasmid DNA (lanes 3-10), digested SLC22A6 CDNA clone (11), AleI (A) or EcoRV (B) digested SLC22A6 positive plasmid DNA (lanes 12-19). Restriction digests with AleI (A) and EcoRV (B) successfully linearised plasmid DNA from 7 of the 8 transformed E.coli colonies included. Colony 5 (lane 16) produced an unexplained band ~3kb. 10kb 3kb 1.5kb 18 10 1 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 19

A

B

107 Figure 4.5: Restriction digest of SLC22A8 positive plasmid DNA from transformed E.coli cultures with AleI (A) and EcoRV (B). Samples were run on a 1% agarose gel using NEB 10kb ladder as reference. From left to right: 1kb DNA ladder (1),

supercoiled SLC22A8 cDNA clone (2), digested SLC22A8 cDNA clone as a positive control (3), supercoiled SLC22A8 positive plasmid DNA (lanes 4,6,8,10,12,14), AleI (A) or EcoRV (B) digested SLC22A8 positive plasmid DNA (lanes 5,7,9,11,13,15). Restriction digests with AleI (A) and EcoRV (B) successfully linearised plasmid DNA from 5 of the 6 transformed E.coli colonies included. Colony 5 (lanes 12-13) produced a double band when digested with AleI (A) and EcoRV (B) suggesting incomplete ligation of the full SLC22A8 template or insertion at the incorrect ligation site.

108 4.2.7MDCK II cell culture

WT MDCK II cells and MDCK II cells stably transfected with SLC22A6 and SLC22A8 were maintained in complete media consisting of DMEM supplemented with 10% FBS and 1% penicillin streptomycin (for stably transfected cells complete media was also supplemented with 0.8µg/ml gentamycin) and kept in an incubator (Thermo Scientific Function Line) at 370_{C with 5% CO}

2. All cell culture was performed in a class II

microbiological safety cabinet. Cells were grown in three T175 flasks at any one time and passaged and seeded to further flasks and/or 12-well plates every 3-4 days, as follows. Briefly, media was removed from each flask using a 25ml serological pipette and each flask washed twice with warm HBSS. A 10ml volume of 0.25% trypsin- EDTA was then added to each flask and flasks were returned to the incubator for 10-15 minutes. Once all cells were detached from the flask, trypsin was neutralised with 20ml of complete media and flask contents transferred to a 50ml Falcon tube before centrifugation at 250 x g for 5 minutes (Thermo Scientific Heraeus Megafuge IIR). The supernatant was removed and the resulting pellet resuspended in 1ml of complete media. Pellet suspensions were combined (three in total) and made up to a total volume of 10ml. If seeding to flasks only 2ml of the resuspended cells were added to 3x T175 flasks to give a 1:5 dilution and allowed to grow on. When seeding to plates, a cell count was first made (as described in section 3.2.4) to give the number of cells per ml and the cell solution subsequently diluted with complete media to a density of 1.5x105 cells per ml. A 1ml aliquot was then added to each well of a 12-well plate.

4.2.8Generation of SLC22A6_MDCK II and SLC22A8_MDCK II cell lines