DNeasy Blood & Tissue Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Downstream tips
- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Publication protocol

Measurement of KSHV genome nuclear delivery by real-time DNA PCR
A pure nuclear fraction was obtained from cells using the Nuclei EZ Prep nuclear isolation kit (Sigma) as per the manufacturer’s instructions. Briefly, HMVEC-d and HUVEC cells were infected with KSHV (30 DNA copies/cell) for 2 h at 37°C, washed twice with HBSS, treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized virus, washed and treated with DNAse I for 10 min at 37°C. Cells were subsequently washed, lysed on ice for 5 min with a mild lysis buffer (Sigma) followed by centrifugation at 500xg for 5 min to obtain the concentrated nuclear pellet. The cytoskeletal components loosely bound to the nuclei were removed by subjecting the nuclear pellet to a second cycle of lysis and centrifugation steps as described previously []. The pure nuclear fraction was then used for DNA isolation followed by amplification of the KSHV ORF73 gene by real-time DNA PCR as described previously [, ] to measure the nuclear entry of KSHV genome.

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Manufacturer protocol

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