AllPrep DNA/RNA Mini 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

PCR and Sequencing
Genomic DNA was extracted from tissue samples and SL-29 cells using the AllPrep dual DNA/RNA extraction kit (QIAGEN). Initial PCR amplification of endogenous retroviral fragments was performed using PCR primers (Integrated DNA Technologies) directed against two highly conserved motifs in retroviral protease (PR) and RT proteins. After initial sequencing of this genomic region, a combination of gene-specific primers and degenerate primers were used to amplify the remaining regions of the REV genomes found in , , and . LTR regions and genomic insertion sites were amplified and cloned by ligation-mediated PCR, using the GenomeWalker Universal kit (Clontech). For complete genome sequencing of DIAV, primers were based on equivalent targets in REV and SNV and were used to amplify multiple overlapping regions of the DIAV genome. A list of primer sequences, the genomes on which they were used, and their coordinates (based on alignment to the DIAV genome) can be found in . Basic PCR conditions were used for almost all reactions (denaturation at 95°C for 2 min, followed by 30 cycles of 94°C for 15 s, 55°C for 30 s, and 68°C for 1 min, final elongation for 10 min), although annealing temperatures and elongation times varied depending on the primers used (details available on request). For all reactions, gel-resolved amplicons were excised from 1% agarose gels and purified using the Qiaquick kit (QIAGEN) before TA cloning into pCR2.1 (Life Technologies, La Jolla, CA) and sequencing. All sequence analysis was performed by the GeneWiz commercial sequencing facility (GeneWiz, South Plainfield, NJ). Sequences obtained in this study have been submitted to Genbank under the following accession numbers: DIAV (KF313137); Echidna-ERV (KF313136), and -ERV (KF313135).



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