Ez1 2 dna tissue kit

Genomic DNA was extracted from tissues with the EZ1&2 DNA Tissue Kit (Qiagen, Valencia, CA, USA) using the EZ1^® Advanced XL instrument (Qiagen) according to the manufacturer’s instructions.
The total concentration of genomic DNA in each sample was evaluated by amplification of the β actin gene, using a standard reference curve, prepared from commercialized mouse DNA (Promega, Madison, WI, USA) ranging from a genome equivalent DNA (gEq) of 70,000 cells (70,000 gEq) to a gEq of 1 cell (1 gEq), with the conversion factor being ~6.6 pg of DNA per cell. Forward 5′-ACTCATCgTACTCCTgCTTgCT-3′ and reverse 5′-AgCTCACCATTCACCATCTTgT-3′ primers and 5′FAM-ATggAgCCACCgATCCACACAgA-3′TMR probe were synthesized by TIB MolBiol (Berlin, Germany) and used at 300 and 100 nM, respectively. Quantitative TaqMan^® PCR was performed using Light Cycler FastStart DNA MasterPLUS reaction kits on a LightCycler^®480 instrument (Roche Diagnostics, Basel, Switzerland). The amplification conditions consisted of an initial incubation at 95°C for 10 min and 45 cycles of 95°C for 15 s and 60°C for 1 min. Data were analyzed using LightCycler software version 3.5.3.

A total of 27 remnant lesion swab specimens were tested. DNA was extracted using the Qiagen EZ1&2 DNA Tissue Kit from specimens that were previously tested with the non-variola Orthopoxvirus real-time PCR assay on the ABI 7500 FastDx instrument following Centers for Disease Control and Prevention Laboratory Response Network protocols [32 (link)]. Libraries were prepared for sequencing using the Illumina DNA prep kit. Pooled libraries were sequenced on the Illumina MiSeq v2 (paired-end 150), with 0.5 to 1 million reads per library.