SspI-HF®

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	Twenty micrograms of each DNA sample was digested overnight at 37°C with either AflII-HF or SspI-HF restriction endonucleases according to the manufacturer's recommendations (New England BioLabs, Ipswich, MA).

Protocol tips
Twenty micrograms of each DNA sample was digested overnight at 37°C with either AflII-HF or SspI-HF restriction endonucleases according to the manufacturer's recommendations (New England BioLabs, Ipswich, MA).

Publication protocol

"Total genomic DNA was purified from rKSHV.294 recombinant or JSC-1 wild-type virus-infected cells by standard methods (46). Twenty micrograms of each DNA sample was digested overnight at 37°C with either AflII-HF or SspI-HF restriction endonucleases according to the manufacturer's recommendations (New England BioLabs, Ipswich, MA). Gel electrophoresis, alkaline transfer, Southern blot hybridization, and stringency washes were performed as described by Sambrook and Russell (46). Prehybridizations and hybridizations were carried out in ULTRAhyb ultrasensitive hybridization buffer (AM8670; Applied Biosystems). To generate biotin-labeled probes, PCRs were carried out as described above except that the starting concentration of dTTP was reduced from 200 to 120 μM, and reaction mixtures were supplemented with 80 μM biotin-16-dUTP (Biotium, Hayward, CA). Five primer sets were used to generate 5 separate KS-specific probes that, together, correspond to the entire wild-type 4,774-bp BamHI KSHV fragment used to construct rKSHV.294. One other primer pair was used to generate a 791-bp biotin-labeled probe corresponding to the neomycin resistance gene, present only within rKSHV.294. Synthesis of individual labeled PCR products was confirmed by gel electrophoresis, and positive detection of biotin incorporation was verified by dot blot analyses of the individual probes after removal of unincorporated label. Hybridization of biotinylated probes to the DNA blots was detected using peroxidase-labeled streptavidin (catalog no. 474-3000; KPL, Gaithersburg, MD) at a 1:500 dilution in 1× PBS-0.05% Tween 20, followed by chemiluminescent substrate (SuperSignal West Pico chemiluminescent substrate, product no. 43080; Thermo Scientific, Rockford, IL) and exposure of X-ray film (CL-XPosure film, product no. 34092; Pierce) according to the manufacturers' recommended protocols.

The primers used to generate the KSHV probes were as follows: KS1F, GTCGGTGTCATGACAAACTG; KS1R, CGACGAAGATAGCACGATAC; KS2F, GCATCATGGATCGCAATGAG; KS2R, CACCGTCAATTATCATGCTC; KS3F, GAGCATGATAATTGACGGTG; KS3R, TTGCGGCGAGGTGCAGTAATTTC; KS4F, TTGCCAAACCCCATGGCAGAGTG; KS4R, CTCGTTTAAAGGCACCAG; KS5F, CTGGTGCCTTTAAACGAG; and KS5R, ATGGCTAGATTTCGCACCAG. Those primers used to generate the Neo probe were neoF, CAAGATGGATTGCACGCAG, and neoR2, CCCGCTCAGAAGAACTCGTC"

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