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3.2.2.1

Extraction of genomic DNA for molecular biological assays

Extraction of genomic DNA from cultured cell lines as well as from murine tissues have been performed with the Qiagen DNeasy Blood & Tissue Kit according to manufacturer’s instructions, with the exception that vortexing was avoided to minimize shearing of genomic DNA.

3.2.2.2

Extraction of genomic DNA from mouse tails for genotyping

VIOLA mice progeny were bred as heterozygotes and were genotyped for the presence of the luc

gene. To this end, 0.5 -1 cm mouse tails pieces were digested o.n. with 500 µl proteinase K buffer supplemented with 19,2 µg proteinase K at 55 °C under constant shaking. The next day, samples were vortexed for 30 s and residual debris removed by centrifugation at 20,000 × g for 2 min at

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RT. The supernatants were transferred into a fresh tube, mixed with 500 µl isopropanol and DNA was pelleted by centrifugation at 20,000 × g for 20 min at RT. Next, the pellet was washed with 500 µl 70 % EtOH to remove salts by another centrifugation for 10 min. Supernatants were aspired, the genomic DNA dried at 37 °C and finally resuspended in 100 µl 10 mM Tris-HCl, pH 7.5. Typically 2 µl of the digest were used for genotyping via PCR (see 3.2.4.2.1).

Proteinase K-Buffer: 200 mM NaCL 100 mM Tris pH 8 5 mM EDTA 1 % (w/v) SDS 3.2.3 Cloning Techniques

3.2.3.1

Restriction enzyme digest

Restriction fragment analysis as well as the preparation of linear DNA fragments was performed with restriction endonucleases. For analytical analysis 1 µg of plasmid DNA was digested with 5 U restriction enzyme for 1 h. Preparative digests 10 µg DNA were digested with 25 U restriction enzyme for 3 h. Temperature and buffers were adjusted according to manufacturer’s instructions. Digestion of genomic DNA was performed with at least 8 U restriction enzyme/µg DNA o.n.

3.2.3.2

DNA precipitation

For concentration or removal of reaction components DNA was precipitated from solution by addition of 1/10 volume 3 M sodium acetate (pH 5.3) and three volumes 100 % ethanol. The solution was mixed and either incubated at -80 °C for 20 min or on ice for 1 h. DNA was subsequently precipitated by centrifugation at 20,000 × g at 4 °C for 30 min. The DNA pellet was washed with 70 % ethanol. After another centrifugation at 20,000 × g at 4 °C for 10 min, the pellet was dried at 45 °C. DNA was subsequently resuspended in 10 mM Tris-HCl, pH 7.5.

3.2.3.3

Blunting of DNA overhangs by Klenow polymerase

For cloning fragments with incompatible restriction sites, fragments were subjected to Klenow Polymerase that fills in 5´ overhangs and removes 3´ overhangs of digested restriction sites to form blunt ends. The reaction was performed in 1 × NEB 2 buffer supplemented with dNTP to a final concentration of 33 µM. One unit Klenow Polymerase per µg DNA was added in a total

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volume of 50 µl. After 15 min incubation at RT, the reaction was stopped by addition of 2 µl 0.5 M EDTA prior to heat inactivation at 75 °C for 20 min. Before using the blunted fragment in further cloning steps, DNA was purified via QIAquick PCR Purification Kit according to manufacturer’s instructions.

3.2.3.4

Dephosphorylation of DNA fragments

To avoid re-circularization of restriction enzyme digested vector DNA, fragments were dephosphorylated with 1 U/µg Antarctic phosphatase for 1 h at 37 °C. The enzyme was inactivated at 65 °C for 10 min and linearized fragments were purified via QIAquick PCR Purification Kit according to manufacturer’s instructions.

3.2.3.5

Purification of DNA from agarose gels

For isolation of DNA fragments from agarose gels (3.2.4.1), fragments were excised on a trans-

illuminator with longwave UV light and purified with the QIAquick Gel Extraction Kit according to manufacturer’s instructions.

3.2.3.6

Ligation of DNA fragments

DNA fragments were ligated using T4 DNA ligase with a molar ratio of 1:3 between vector (100 ng) and insert. Ligation was performed in a total reaction volume of 20 µl with 400 U T4 DNA ligase in 1× T4 DNA Ligase buffer in a water bath at 16 °C o.n. In control reactions, insert fragments were replaced with the same volume water. Typically 50 µl electrocompetent bacteria were transformed with 4 µl of the ligation reaction.

3.2.3.7

Manipulation of bacterial artificial chromosomes by homologous recombination

Due to the big size of BACs coding for the entire herpesviral genomes, standard cloning procedures are not feasible [66]. To generate herpesviral mutants, homologous recombination of linear fragments with the BAC DNA was applied according to the protocol established by Wagner and colleagues [129].

In this work, a kanamycin selection cassette was inserted next to the origin of replication (see Figure 15) by homologous recombination. To this end, a linear fragment (containing a kanamycin selection marker, the bacterial origin of replication R6K and 40 bp sequences homologues to the viral genome on both sites) was amplified from the plasmid p06kan by PCR with the primer H3-MCMVori-ori6kan-for and H5-MCMVori-ori6kan-rev. The fragment was purified by DNA precipitation (see 3.2.3.2) and electrocompetent L-Arabinose induced DH10B bacteria harbouring pSM3fr and pKD46 were transformed with 1.5 µg. By induction with L-

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Arabinose, recombinases were expressed in a controlled manner and mediated the recombination of the linear fragment and the homologous region on the BAC during 2 h incubation at 37 °C. Bacteria containing an insertion of the kanamycin cassette were selected by growing on LB- CAM-KAN-agar plates. Correct insertion was confirmed by RFLP analysis of the candidate pSM3fr-ori6kan BAC clones.