GUILAIN A LA CORTE

2.2.2.1Plasmid DNA isolation

For most plasmids, plasmid DNA was isolated from 1.5 to 4 mL of liquid E. coli cultures using the Axygen plasmid MiniPrep kit according to the manufacturer’s instructions, including the optional W2 washing step. The DNA was eluted in 60 μL of sterile water. If a plasmid was low copy number, the transformed bacteria grew poorly or a large mass of DNA was required, DNA was extracted from 100 mL of liquid E. coli cultures using the Axygen MidiPrep kit according to the manufacturer’s instructions. DNA was eluted in 500 μL of sterile water.

2.2.2.2Purification of PCR products

PCR products were purified using the Gene Clean Spin kit according to the manufacturer’s instructions. The DNA was eluted in 15 μL of sterile water.

In some circumstances, DNA was purified from an agarose gel to ensure that DNA of the correct size was isolated. After DNA gel electrophoresis (refer to Section 2.2.5), the agarose gel was stained with DNA staining solution and illuminated using an Invitrogen Safe Imager Blue-Light Transilluminator (Life Technologies). The bands containing the DNA fragment of the appropriate size were excised from the gel. The Gene Clean Spin kit was used to purify the DNA fragments from the gel according to the manufacturer’s instructions. The DNA was eluted in 15 μL of sterile water.

2.2.2.3Crude plasmid isolation for size differentiation (colony cracking)

Colony cracking was used to rapidly screen transformed bacterial colonies to identify clones carrying a plasmid of the correct size. This method was used when the insert was large and there was a substantial size difference between the parent vector and the newly constructed plasmid. Single bacterial colonies were selected from LB-agar plates and spotted onto a fresh LB-agar plate with the appropriate antibiotic, with the remainder resuspended in 40 μL 10 mM EDTA. Next, 50 μL of freshly made cracking buffer was added to each colony and incubated for five minutes at room temperature. Then, 10 μL of marker mix was added and incubated for five minutes on ice. The mixture was centrifuged at 20200 g for three minutes, and approximately 30 μL of the supernatant was loaded onto a 1% (w/v) agarose gel and run against the supercoiled DNA ladder (refer to Section 2.2.5).

2.2.2.4Purification of infectious HSV-1 DNA for transfection

To generate infectious HSV-1 DNA for the cotransfection of viral and plasmid DNA to construct recombinant viruses (refer to Section 2.2.11.1), confluent Vero cell monolayers were infected at a multiplicity of infection (MOI) of 0.1 and incubated at 37°C and 5% CO2 for 24 hours or until full CPE is evident. The cells were harvested in media and centrifuged at 820 g for 10 minutes at 4°C and the supernatant was discarded. The cell pellet was washed with cold PBS, before being resuspended in TE with 0.5% (w/v) SDS and 50 μg/mL proteinase K, and incubated at 37°C overnight. This aqueous mix was then mixed with an equal volume of phenol and inverted carefully to mix the two phases. This mix was then centrifuged at 4300 g for 15 minutes to separate the two phases. The aqueous phase was then re-extracted with phenol:chloroform at least twice more. The final aqueous phase was then extracted with chloroform to remove trace phenol, before the DNA is precipitated by mixing the last aqueous phase with 0.1 volume of 3 M sodium acetate and three volumes of 100% ethanol. The DNA was pelleted by centrifugation at 5050 g for five minutes. The pellet was gently washed with 70% ethanol before being air dried and then suspended in an appropriate volume of TE and incubated on ice overnight.

2.2.2.5Crude virus DNA preparation from isolated plaques and viral stocks

Viral DNA was prepared from isolated plaques to serve as the template for diagnostic PCRs (refer to Section 2.2.12). 96 well plates of confluent Vero cells were prepared and 75 µL of the virus from individual isolated plaques was added to each well as appropriate. The cells were incubated for two days at 37°C in 5% CO2. The media was then removed and the infected cell monolayers were washed with PBS before 100 μL of 10 μg/mL proteinase K in 1× ThermoPol buffer in sterile water was added to each well. The plates were then

frozen in a -80°C freezer and thawed to lyse cell membranes to release the virus. Alternatively, 10 μL of virus was mixed with 1× ThermoPol buffer in sterile water to a total volume of 100 μL. The plates were incubated at 56°C for 20 minutes, and then 85°C for 15 minutes. The undiluted supernatant was used as template for diagnostic PCRs.

2.2.2.6 Purification of HSV-1 DNA for use in whole genome digests

To generate purified HSV-1 DNA for use in whole genome digests (refer to Section 2.2.4), confluent Vero cell monolayers were infected at an MOI of 0.1 and incubated at 37°C and 5% CO2 for 24 hours or until full CPE is evident. The cells were harvested in media and centrifuged at 820 g for 10 minutes at 4°C to separate the supernatant and cellular debris. The supernatant was collected and stored on ice. The cells were then lysed with RSB buffer containing 0.5% (v/v) IGEPAL by incubation for ten minutes on ice. The nuclei are then pelleted by centrifuging this sample at 820 g for 10 minutes at 4°C. The supernatant was collected and the cell pellet was extracted again using RSB buffer containing 0.5% (v/v) IGEPAL. All of the supernatants were pooled and pelleted by centrifuging at 17 680 g

for two hours at 4°C. The pellet was then resuspended in 1.6 mL of TE with 0.5% SDS and 50 μg/mL proteinase K, and incubated at 37°C for 5 minutes. This aqueous mixture was divided into two separate aliquots and was then mixed with an equal volume of phenol:chloroform and carefully inverted until all phases were uniformly mixed. This mix was then centrifuged at 20200 g for 10 minutes to separate the two phases. The aqueous phase was then re-extracted with phenol:chloroform at least twice more. The DNA was precipitated by mixing the last aqueous phase with 0.1 volume of 3M sodium acetate and three volumes of 100% ethanol. The DNA was pelleted by centrifugation at 20200 g for 20 minutes. The pellet was gently washed with 70% ethanol before being air dried and then resuspended in an appropriate volume of TE and incubated on ice overnight.

2.2.2.7Purification of nucleic acids by sodium acetate/ethanol precipitation

To purify linearised plasmids prior to transfection (refer to Section 2.2.11), an ethanol/sodium acetate precipitation was performed to purify the DNA in a sterile environment. The DNA was precipitated by the addition of 0.1 volume of 3 M sodium acetate and three volumes of 100% ethanol and vigorous mixing. If the DNA did not immediately form a visible precipitate, it was left to incubate on ice for 15 minutes. The DNA was centrifuged at 20200 g for 20 minutes to pellet the DNA, and then washed with 70% ethanol. The ethanol was removed in a clean environment within a biosafety cabinet and allowed to air dry for 15 minutes. The DNA was then resuspended in an appropriate volume of sterile DNA.