DNA extraction

For all nymph and adult tick samples DNA extraction was carried out as described by Gern et al (2010). Briefly, each tick was placed into an individual 1.5 ml Surelock microcentrifuge tube (Starlabs, Milton Keynes) containing 100µl (un-engorged nymph), 200µl (engorged nymphs or un-engorged adults), 500µl (engorged adults) of 1.25 % ammonium hydroxide (Sigma, Dorset). The tick was ground using a sterile pipette tip then the tube was closed, locked and placed onto a heating block at 100o_{C for 20 min. Each tube was briefly}

centrifuged then returned to the heating block for a further 10 min, with the lid open, to evaporate off half of the solution. Finally, each tube was closed, relocked and stored at -20o_{C until required. In order to control for cross-contamination between samples, cross-} contamination controls were included every time DNA extractions were prepared. These comprised of tubes containing only 100µl, 200µl or 500µl of 1.25 % ammonium hydroxide that were co-processed with tubes containing ticks. One control was used for every four ticks processed. If the ticks were heavily engorged, one in ten dilutions of the DNA extracts was made for engorged nymphs and one in a hundred for engorged adults to prevent any inhibition during PCR. DNA was not quantified after each extraction due to the volume of samples that were processed

For larvae samples, DNA extraction was carried out using a DNeasy extraction kit (Qiagen, Hilden Germany), using the method for purification of total DNA from ticks (Qiagen, Hilden Germany). Up to 10 larvae were pooled for DNA extraction. Only larvae from the same squirrel carcass were pooled together, resulting in pools from 1 to 10 larvae. Up to 10 larvae were placed in a Sure lock microcentrifuge tube (Starlabs, Milton Keynes) with 180µl of buffer ATL. The larvae were homogenised using a sterile pipette tip. 20µl of proteinase K was added and then the lids closed, all samples were thoroughly vortexed and then placed into a water bath at 56ºC overnight. The samples were then vortexed for 15 seconds and 200µl of AL buffer was added to the sample and vortexed again. The samples were

incubated at 70ºC for 10 mins. 1µl of carrier RNA was added to the samples and vortexed, after which 230µl of ethanol (96-100%) was added and then again vortexed.

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This mixture was transferred from the microcentrifuge tube into a DNeasy mini spin column which was placed into a 2ml collection tube. This was centrifuged at 6,000 x g for 1 min and the flow through was discarded. 500µl of buffer AW1 was added to the spin column and it was centrifuged again for another min at 6,000 x g, the flow through was discarded. 500µl of buffer AW2 was added to the spin column and it was centrifuged for 3 mins at 20,000 x g, the flow through was discarded. The spin column was placed into a 1.5ml sure lock

microcentrifuge tube (Starlabs, Milton Keynes), 35µl of AE buffer was added to the column, directly on top of the membrane and left to incubate at room temperature for 1 min before being centrifuged for 1 min at 6,000 x g to elute, another 30µl of AE buffer was then added to the spin column and the incubation and centrifugation step repeated to have a final eluate of 60µl. The eluent was analysed on the Nano drop 2000 Spectrophotometer (Thermoscientific, USA) to obtain the nucleic acid concentration and to assess if the extraction had worked. The eluent was kept at -20º until testing.

2.3.2 Tissue samples

DNA extraction of squirrel ear biopsy punches was carried out using an Isolate II Genomic DNA kit (Bioline, London). For each squirrel five biopsy punches from each ear were combined. Extraction controls were made every ten samples. The 10 biopsy punches were place in a Surelock microcentrifuge tube (Starlabs, Milton Keynes) with 180 µl of Lysis buffer GL and 25µl of proteinase K, then were thoroughly vortexed and placed into a water bath at 56ºC overnight. The samples were vortexed for 15 seconds and 200µl of Lysis buffer G3 was added and vortexed. The samples were then incubated at 70ºC for 10 mins. Then 210µl of ethanol (96-100%) was added and vortexed.

This mixture was transferred from the microcentrifuge tube into an Isolate II spin column that was placed into a 2ml collection tube. This was centrifuged at 11, 000 x g for 1 min and the flow through was discarded. 500µl of buffer GW1 was added to the spin column and it was centrifuged for a further min at 11,000 x g. The flow through was again discarded. 600µl of buffer AW2 was added to the spin column and it was centrifuged for 1 min at 11,000 x g, the flow through was discarded. To dry the silica membrane the spin column was

centrifuged at 11,000 x g for 1 min to remove any residual ethanol. The spin column was then placed into a 1.5ml Sure lock microcentrifuge tube (Starlabs, Milton Keynes), 100µl of

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elution buffer which has been preheated to 70ºC was added to the column, directly on top of the membrane and left to incubate at room temperature for 1 min and then centrifuged for 1 min at 11,000 x g to elute. The eluent was analysed on the Nano drop 2000

Spectrophotometer (Thermoscientific, USA) to obtain the nucleic acid concentration and to assess if the extraction had worked. The eluent was kept at -20º until testing.